Humanized antibodies

ABSTRACT

Disclosed herein are humanized antibodies in which human germline residues are introduces to the complementarity determining regions (CDRs) of a non-human donor antibody. Also described herein are libraries of antibody variable domains (e.g., phage-display libraries) and methods for screening for humanized antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.62/162,905, filed May 18, 2016, which is incorporated by reference inits entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 16, 2016, is namedPC72222A_Seq_Listing_ST25.txt and is 367,747 bytes in size.

FIELD OF THE INVENTION

This invention relates to antibody humanization by introducing humangerm-line residues in complementarity determining regions (CDRs).

BACKGROUND OF THE INVENTION

Monoclonal antibodies can rapidly be produced by the mouse immune systemfor biological studies. In a clinical setting, however, the use of thesemurine antibodies can result in a human anti-mouse antibody response(HAMA). Chimeric antibodies can reduce anti-IgG responses in human, butmurine v-domains may still have provocative T-cell epitope content,necessitating “humanization” of their framework regions.

Classical humanization generally begins by transferring all six murinecomplementarity determining regions (CDRs) onto a human antibodyframework (Jones et al., Nature 321, 522-525 (1986)). These CDR-graftedantibodies generally do not retain their original affinity for antigenbinding, and in fact, affinity is often severely impaired. Besides theCDRs, certain non-human framework residues must also be incorporatedinto the variable domains to maintain proper CDR conformation (Chothiaet al., Nature 342:877 (1989)). The incorporation of murine residues atkey positions in the human frameworks to restore function is generallyreferred to as “back-mutations.” Back-mutations can support structuralconformation of the grafted CDRs and restore antigen binding andaffinity. Many of the framework positions that are likely to affectaffinity have been identified, thus structural modeling to select newresidues in a stepwise fashion can generally lead to variants withrestored antigen binding. Alternatively, phage antibody librariestargeted at these residues can also be used to enhance and speed up theaffinity maturation process (Wu et al., J. Mol. Biol. 294:151-162 (1999)and Wu, H., Methods in Mol. Biol. 207:197-212 (2003)).

Current humanization techniques still suffer from flaws, such as highnon-human amino acid content retention; grafting into poorly understoodframeworks; requirement for homology modeling of the v-domains, which isoften inaccurate; or a co-crystal structure with the target antigen.Therefore, there is a need to develop new humanization methods.

SUMMARY OF THE INVENTION

Disclosed herein are “ultra” humanized antibodies in which humangermline residues are introduced to the complementarity determiningregions (CDRs) of a non-human donor antibody. Also described herein arelibraries and methods for screening for humanized antibodies.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments (E).

E1. A method of generating a library comprising a plurality ofpolypeptides, for selection of a humanized antibody that binds to atarget antigen, comprising:

-   -   (a) obtaining the sequence a non-human donor antibody that binds        to said target antigen, and determining the donor CDR-L1,        CDR-L2, CDR-L3, CDR-H1, and CDR-H2 sequences of said non-human        antibody;    -   (b) obtaining the sequences of a human germline VL and a human        germline VH, and determining the germline framework and germline        CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 sequences of said        human VL and VH;    -   (c) aligning each of the non-human donor CDR-L1, CDR-L2, and        CDR-L3 sequences with the corresponding germline CDR sequence        from said human VL, and each of the non-human CDR-H1 and CDR-H2        sequences with corresponding germline CDR sequence from said        human VH;    -   (d) identifying positions in CDR-L1, CDR-L2, CDR-L3, CDR-H1, and        CDR-H2 where human germline residue is the same as, or different        from, the corresponding non-human donor residue;    -   (e) generating a library of polypeptides, each polypeptide        comprising an antibody variable domain, wherein said antibody        variable domain comprises (1) a VH domain comprising: the        framework sequence of the human germline VH from step (b), and        CDR-H1, CDR-H2, and CDR-H3; and (2) a VL domain comprising: the        framework sequence of the human germline VL from step (b), and        CDR-L1, CDR-L2, and CDR-L3; wherein:        -   (i) for each individual position within CDR-L1, CDR-L2,            CDR-L3, CDR-H1 and CDR-H2:            -   if the human germline residue at said position is the                same as the corresponding non-human donor residue, all                polypeptides in the library comprise the human germline                residue at said position;            -   if the human germline residue at the position is                different from the corresponding non-human donor                residue, a portion of the polypeptides in the library                comprise the human germline residue at said position,                the remainder of the polypeptides comprise the                corresponding non-human donor residue at said position;        -   (ii) for each individual position within CDR-H3, the residue            is any one of the 20 natural amino acid residues;        -   (iii) less than 1% of the polypeptides in said library            comprise the original non-human donor CDR-L1, CDR-L2,            CDR-L3, CDR-H1, and CDR-H2 sequences; and        -   (iv) less than 1% of the polypeptides in said library            comprise the original human VL germline CDR-L1, CDR-L2, and            CDR-L3, and the original human VH germline CDR-H1 and CDR-H2            sequences.            E2. The method of embodiment 1, wherein for each individual            position within CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2,            if the human germline residue and non-human donor residue            are different according to step (d), the percentage of            polypeptides comprising the human germline residue at said            position is from about 5% to about 95%, the remainder            comprising the corresponding non-human donor residue at said            position.            E3. The method of embodiment 1 or 2, wherein for each            individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,            and CDR-H2, if the human germline residue and non-human            donor residue are different according to step (d), the            percentage of polypeptides comprising the human germline            residue at said position is from about 10% to about 90%, the            remainder comprising the corresponding non-human donor            residue at said position.            E4. The method of any one of embodiments 1-3, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 15% to about 85%, the            remainder comprising the corresponding non-human donor            residue at said position.            E5. The method of any one of embodiments 1-4, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 20% to about 80%, the            remainder comprising the corresponding non-human donor            residue at said position.            E6. The method of any one of embodiments 1-5, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 25% to about 75%, the            remainder comprising the corresponding non-human donor            residue at said position.            E7. The method of any one of embodiments 1-6, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 30% to about 70%, the            remainder comprising the corresponding non-human donor            residue at said position.            E8. The method of any one of embodiments 1-7, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 35% to about 65%, the            remainder comprising the corresponding non-human donor            residue at said position.            E9. The method of any one of embodiments 1-8, wherein for            each individual position within CDR-L1, CDR-L2, CDR-L3,            CDR-H1, and CDR-H2, if the human germline residue and            non-human donor residue are different according to step (d),            the percentage of polypeptides comprising the human germline            residue at said position is from about 40% to about 60%, the            remainder comprising the corresponding non-human donor            residue at said position.            E10. The method of any one of embodiments 1-9, wherein for            each individual position within CDR-H3, each of the 20            natural amino acid residues is represented by at least about            0.1% of the polypeptides in the library.            E11. The method of any one of embodiments 1-10, wherein for            each individual position within CDR-H3, each of the 20            natural amino acid residues is represented by at least about            1% of the polypeptides in the library.            E12. The method of any one of embodiments 1-11, wherein for            each individual position within CDR-H3, each of the 20            natural amino acid residues is represented by at least about            2% of the polypeptides in the library.            E13. The method of any one of embodiments 1-12, wherein for            each individual position within CDR-H3, each of the 20            natural amino acid residues is represented by from about 2%            to about 8% of the polypeptides in the library.            E14. A method of humanizing a non-human donor antibody,            wherein said non-human donor antibody binds to a target            antigen, comprising:    -   (a) obtaining the sequence said non-human donor antibody, and        determining the donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,        and CDR-H3 sequences;    -   (b) obtaining the sequences of a human germline VL and a human        germline VH, and determining the germline framework and germline        CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 sequences of said        human VL and VH;    -   (c) aligning each of the non-human donor CDR-L1, CDR-L2, and        CDR-L3 sequences with the corresponding germline CDR sequence        from said human VL, and each of the non-human CDR-H1 and CDR-H2        sequences with corresponding germline CDR sequence from said        human VH;    -   (d) identifying positions in CDR-L1, CDR-L2, CDR-L3, CDR-H1, and        CDR-H2 where human germline residue is or different from the        corresponding non-human donor residue;    -   (e) obtaining a humanized antibody by:        -   (i) grafting said non-human donor CDR-L1, CDR-L2, and CDR-L3            into said human VL germline framework, and said non-human            donor CDR-H1, CDR-H2, CDR-H3 into said human VH germline            framework; and        -   (ii) further introducing a mutation in at least one, but not            all, of the positions identified in (d), wherein said            mutation comprises replacing a non-human donor residue with            the corresponding human germline residue;        -   (iii) introducing at least one random mutation in CDR-H3,            wherein said mutation comprises replacing a non-human donor            residue with a different amino acid residue;    -   (f) assessing the binding affinity of the humanized antibody        obtained in (e) to the target antigen.        E15. The method of embodiment 14, comprising introducing        mutations in at least three, but not all, of the positions        identified in (d), wherein said mutations comprise replacing a        non-human donor residue with the corresponding human germline        residue.        E16. The method of embodiment 14 or 15, comprising introducing        mutations in at least five, but not all, of the positions        identified in (d), wherein said mutations comprise replacing a        non-human donor residue with the corresponding human germline        residue.        E17. The method of any one of embodiments 14-16, comprising        introducing two or more random mutations in CDR-H3, wherein said        mutations comprise replacing a non-human donor residue with a        different amino acid residue.        E18. The method of any one of embodiments 1-17, wherein said        human germline VH framework sequence comprises a VH3 framework        sequence.        E19. The method of any one of embodiments 1-17, wherein said        human germline VH framework sequence comprises a VH1 framework        sequence.        E20. The method of any one of embodiments 1-17, wherein said        human germline VH framework sequence comprises a VH5 framework        sequence.        E21. The method of any one of embodiments 1-17, wherein said        human germline VH framework sequence comprises the VH framework        sequence of any one of the human germline sequences listed in        Table 2.        E22. The method of any one of embodiments 1-17 and 21, wherein        said human germline VH framework sequence comprises the VH        framework sequence of human germline IGHV3-23 or IGHV1-69.        E23. The method of any one of embodiments 1-17 and 21, wherein        said human germline VH framework sequence comprises the VH        framework sequence of human germline IGHV3-7.        E24. The method of any one of embodiments 1-17, wherein said        human germline VH framework sequence comprises a VH germline        consensus framework sequence.        E25. The method of embodiment 24, wherein said human germline VH        framework sequence comprises the VH framework sequence of any        one of the consensus sequences listed in Table 5.        E26. The method of any one of embodiments 1-25, wherein said        human germline VL framework sequence comprises a V_(K) framework        sequence.        E27. The method of any one of embodiments 1-26, wherein said        human germline VL framework sequence comprises the VL framework        sequence of any one of the human germline sequences listed in        Table 3.        E28. The method of any one of embodiments 1-25 wherein said        human germline VL framework sequence comprises a V_(λ) framework        sequence.        E29. The method of any one of embodiments 1-25 and 28, wherein        said human germline VL framework sequence comprises the VL        framework sequence of any one of the human germline sequences        listed in Table 4.        E30. The method of any one of embodiments 1-25, wherein said        human germline VL framework sequence comprises the VL framework        sequence of human germline IGKV3-20.        E31. The method of any one of embodiments 1-25, wherein said        human germline VL framework sequence comprises the VL framework        sequence of human germline IGKV1-39.        E32. The method of any one of embodiments 1-25, wherein said        human germline VL framework sequence comprises a VL germline        consensus framework sequence.        E33. The method of any one of embodiments 1-25 and 32, wherein        said human germline VL framework sequence comprises the VL        framework sequence of any one of consensus sequences listed in        Table 6.        E34. The method of any one of embodiments 1-33, wherein said        non-human donor antibody is a non-human mammalian antibody.        E35. The method of any one of embodiments 1-34, wherein said        non-human donor antibody is a murine antibody, a rat antibody,        or a rabbit antibody.        E36. The method of any one of embodiments 1-35, wherein said        non-human donor antibody is a murine antibody.        E37. The method of any one of embodiments 34-36, wherein said        human germline VL framework comprises no more than 3        back-mutations or random mutations.        E38. The method of any one of embodiments 34-37, wherein said        human germline VH framework comprises no more than 3        back-mutations or random mutations.        E39. The method of any one of embodiments 34-38, wherein said        human germline VL framework and VH framework together comprise        no more than 3 back-mutations or random mutations.        E40. The method of any one of embodiments 34-39, wherein at        least one back-mutation or random mutation is located between        heavy chain residues H71 to H80.        E41. The method of any one of embodiments 34-39, wherein said        human germline VH framework and human germline VH framework do        not comprise a back-mutation.        E42. The method of any one of embodiments 1-33, wherein said        non-human donor antibody is an avian antibody.        E43. The method of embodiment 42, wherein said human germline VL        framework comprises no more than 5 back-mutations or random        mutations.        E44. The method of embodiment 42 or 43, wherein said human        germline VH framework comprises no more than 5 back-mutations or        random mutations.        E45. The method of any one of embodiments 42-44, wherein said        human germline VL framework and VH framework together comprise        no more than 5 back-mutations or random mutations.        E46. The method of any one of embodiments 42-45, wherein at        least one back-mutation or random mutation is located between        heavy chain residues H71 to H80.        E47. The method of any one of embodiments 42-45, wherein said        human germline VL framework comprises a single back-mutation.        E48. The method of any one of embodiments 42-46, wherein said        human germline VH framework comprises a single back-mutation.        E49. The method of any one of embodiments 42-47, wherein said        non-human donor antibody is a chicken antibody.        E50. The method of any one of embodiments 42-45 and 47-49,        wherein said human germline VL framework comprises a        back-mutation at position 46 (such as Leu46Thr (L46T)).        E51. The method of any one of embodiments 1-13 and 18-50,        wherein said library is a phage display library.        E52. The method of any one of embodiments 1-51, further        comprising: obtaining the sequence of a human germline VH        by: (i) aligning the framework sequence of said non-human donor        antibody VH against a plurality of human germline VH framework        sequences; and (ii) selecting a human germline VH framework that        has the highest sequence identity according to step (i).        E53. The method of any one of embodiments 1-52, further        comprising: obtaining the sequence of a human germline VL        by: (i) aligning the framework sequence of said non-human donor        antibody VL against a plurality of human germline VL framework        sequences; and (ii) selecting a human germline VL framework that        has the highest sequence identity according to step (i).        E54. A library for humanizing a non-human donor antibody that        binds to a target antigen, wherein:    -   (a) said library comprises a plurality of polypeptides, each        polypeptide comprising an antibody variable domain;    -   (b) said antibody variable domain comprises (i) a VH domain        comprising: a human germline VH framework sequence, and CDR-H1,        CDR-H2, and CDR-H3;    -   and (ii) a VL domain comprising: a human germline VL framework        sequence, and CDR-L1, CDR-L2, and CDR-L3;    -   (c) for each individual position within said CDR-L1, CDR-L2,        CDR-L3, CDR-H1, and CDR-H2,        -   if the human germline residue at said position is the same            as the corresponding non-human donor residue, all            polypeptides in the library comprise the human germline            residue at said position;        -   if the human germline residue at the position is different            from the corresponding non-human donor residue, a portion of            the polypeptides in the library comprise the human germline            residue at said position, the remainder of the polypeptides            comprise the corresponding non-human donor residue at said            position;    -   (d) for each individual position within CDR-H3, the residue is        any one of the natural amino acid residues;    -   (e) less than 1% of the polypeptides in said library comprise        the original non-human donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, and        CDR-H2 sequences; and    -   (f) less than 1% of the polypeptides in said library comprise        the original human VL germline CDR-L1, CDR-L2, and CDR-L3, and        the original human VH germline CDR-H1 and CDR-H2 sequences.        E55. The library of embodiment 54, wherein for each individual        position within CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, if        the human germline residue and non-human donor residue are        different at said position, the percentage of polypeptides        comprising the human germline residue at said position is from        about 5% to about 95%, the remainder comprising the        corresponding non-human donor residue at said position.        E56. The library of embodiment 54 or 55, wherein for each        individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1, and        CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 10% to about 90%, the remainder        comprising the corresponding non-human donor residue at said        position.        E57. The library of any one of embodiments 54-56, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 15% to about 85%, the remainder        comprising the corresponding non-human donor residue at said        position.        E58. The library of any one of embodiments 54-57, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 20% to about 80%, the remainder        comprising the corresponding non-human donor residue at said        position.        E59. The library of any one of embodiments 54-58, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 25% to about 75%, the remainder        comprising the corresponding non-human donor residue at said        position.        E60. The library of any one of embodiments 54-59, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 30% to about 70%, the remainder        comprising the corresponding non-human donor residue at said        position.        E61. The library of any one of embodiments 54-60, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 35% to about 65%, the remainder        comprising the corresponding non-human donor residue at said        position.        E62. The library of any one of embodiments 54-61, wherein for        each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1,        and CDR-H2, if the human germline residue and non-human donor        residue are different at said position, the percentage of        polypeptides comprising the human germline residue at said        position is from about 40% to about 60%, the remainder        comprising the corresponding non-human donor residue at said        position.        E63. The library of any one of embodiments 54-62, wherein for        each individual position within CDR-H3, each of the 20 natural        amino acid residues is represented by at least about 0.1% of the        polypeptides in the library.        E64. The library of any one of embodiments 54-63, wherein for        each individual position within CDR-H3, each of the 20 natural        amino acid residues is represented by at least about 1% of the        polypeptides in the library.        E65. The library of any one of embodiments 54-64, wherein for        each individual position within CDR-H3, each of the 20 natural        amino acid residues is represented by at least about 2% of the        polypeptides in the library.        E66. The library of any one of embodiments 54-65, wherein for        each individual position within CDR-H3, each of the 20 natural        amino acid residues is represented by from about 2% to about 8%        of the polypeptides in the library.        E67. The library of any one of embodiments 54-66, wherein said        human germline VH framework sequence comprises a VH3 framework        sequence.        E68. The library of any one of embodiments 54-66, wherein said        human germline VH framework sequence comprises a VH1 framework        sequence.        E69. The library of any one of embodiments 54-66, wherein said        human germline VH framework sequence comprises a VH5 framework        sequence.        E70. The library of any one of embodiments 54-66, wherein said        human germline VH framework sequence comprises the VH framework        sequence of any one of the human germline sequences listed in        Table 2.        E71. The library of any one of embodiments 54-66 and 70, wherein        said human germline VH framework sequence comprises the VH        framework sequence of human germline IGHV3-23 or IGHV1-69.        E72. The library of any one of embodiments 54-66 and 70, wherein        said human germline VH framework sequence comprises the VH        framework sequence of human germline IGHV3-7.        E73. The library of any one of embodiments 54-66, wherein said        human germline VH framework sequence comprises a VH germline        consensus framework sequence.        E74. The library of embodiment 73, wherein said human germline        VH framework sequence comprises the VH framework sequence of any        one of the consensus sequences listed in Table 5.        E75. The library of any one of embodiments 54-74, wherein said        human germline VL framework sequence comprises a V_(K) framework        sequence.        E76. The library of any one of embodiments 54-75, wherein said        human germline VL framework sequence comprises the VL framework        sequence of any one of the human germline sequences listed in        Table 3.        E77. The library of any one of embodiments 54-74, wherein said        human germline VL framework sequence comprises a V_(λ) framework        sequence.        E78. The library of any one of embodiments 54-74 and 77, wherein        said human germline VL framework sequence comprises the VL        framework sequence of any one of the human germline sequences        listed in Table 4.        E79. The library of any one of embodiments 54-74, wherein said        human germline VL framework sequence comprises the VL framework        sequence of human germline IGKV3-20.        E80. The library of any one of embodiments 54-74, wherein said        human germline VL framework sequence comprises the VL framework        sequence of human germline IGKV1-39.        E81. The library of any one of embodiments 54-74, wherein said        human germline VL framework sequence comprises a VL germline        consensus framework sequence. E82. The library of any one of        embodiments 54-74 and 81, wherein said human germline VL        framework sequence comprises the VL framework sequence of any        one of consensus sequences listed in Table 6.        E83. The library of any one of embodiments 54-82, wherein said        non-human donor antibody is a non-human mammalian antibody.        E84. The library of any one of embodiments 54-83, wherein said        non-human donor antibody is a murine antibody, a rat antibody,        or a rabbit antibody.        E85. The library of any one of embodiments 54-84, wherein said        non-human donor antibody is a murine antibody.        E86. The library of any one of embodiments 83-85, wherein said        human germline VL framework comprises no more than 3        back-mutations or random mutations.        E87. The library of any one of embodiments 83-86, wherein said        human germline VH framework comprises no more than 3        back-mutations or random mutations.        E88. The library of any one of embodiments 83-87, wherein said        human germline VL framework and VH framework together comprise        no more than 3 back-mutations or random mutations.        E89. The library of any one of embodiments 83-88, wherein at        least one back-mutation or random mutation is located between        heavy chain residues H71 to H80.        E90. The library of any one of embodiments 83-88, wherein said        human germline VH framework and human germline VH framework do        not comprise a back-mutation.        E91. The library of any one of embodiments 54-82, wherein said        non-human donor antibody is an avian antibody.        E92. The library of embodiment 91, wherein said human germline        VL framework comprises no more than 5 back-mutations or random        mutations.        E93. The library of embodiment 91 or 92, wherein said human        germline VH framework comprises no more than 5 back-mutations or        random mutations.        E94. The library of any one of embodiments 91-93, wherein said        human germline VL framework and VH framework together comprise        no more than 5 back-mutations or random mutations.        E95. The library of any one of embodiments 91-94, wherein at        least one back-mutation or random mutation is located between        heavy chain residues H71 to H80.        E96. The library of any one of embodiments 91-95, wherein said        human germline VL framework comprises a single back-mutation.        E97. The library of any one of embodiments 91-96, wherein said        human germline VH framework comprises a single back-mutation.        E98. The library of any one of embodiments 91-97, wherein said        non-human donor antibody is a chicken antibody.        E99. The library of any one of embodiments 91-94 and 96-98,        wherein said human germline VL framework comprises a        back-mutation at position 46 (such as Leu46Thr (L46T).        E100. The library of any one of embodiments 54-99, wherein said        library is a phage display library.        E101. A plurality of nucleic acid molecules encoding the library        of any one of embodiments 54-100.        E102. The plurality of nucleic acid molecules of embodiment 101,        wherein said nucleic acid molecules are phagemid vectors.        E103. A plurality of host cells comprising the nucleic acid        molecules of embodiment 101 or 102.        E104. A method of identifying an antibody variable domain        sequence that binds to a target antigen, comprising:    -   (a) obtaining the library of any one of embodiments 54-100;    -   (b) selecting a polypeptide that binds to said target antigen,        with an affinity (Kd) value of 5×10⁻⁵ M or less, and    -   (c) obtaining the sequence of the antibody variable domain of        the polypeptide selected in step (b).        E105. The method of embodiment 104, wherein said an antibody        variable domain binds to said target antigen with a binding        affinity (Kd) value that is equal or less than that of said        non-human donor antibody.        E106. The method of embodiment 104 or 105, further comprising        producing a nucleic acid vector encoding a polypeptide        comprising the antibody variable domain selected from step (c).        E107. The method of embodiment 106, further comprising        introducing said vector into a host cell.        E108. The method of embodiment 106 or 107, wherein said vector        is an expression vector.        E109. The method of embodiment 107 or 108, further comprising        culturing said host cell in a medium under a suitable condition,        such that said polypeptide is expressed. E110. The method of        embodiment 109, further comprising harvesting said polypeptide        from the medium.        E111. The method of embodiment 110, further comprising purifying        said polypeptide.        E112. A humanized antibody or antigen-binding fragment thereof        that binds to a target antigen, wherein:    -   (a) said antibody or antigen-binding fragment thereof        comprises (i) a VH domain comprising: a human germline VH        framework sequence, and CDR-H1, CDR-H2, and CDR-H3; and (ii) a        VL domain comprising: a human germline VL framework sequence,        and CDR-L1, CDR-L2, and CDR-L3;    -   (b) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 are derived        from corresponding CDRs from a non-human donor antibody that        binds to said target antigen;    -   (c) for each position within said CDR-L1, CDR-L2, CDR-L3,        CDR-H1, and CDR-H2, the residue is either human germline residue        from said human germline VL or VH, or corresponding residue from        said non-human donor antibody;    -   (d) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each        comprises at least one more human germline residue as compared        to the corresponding non-human donor CDR,    -   (e) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each        comprises at least one more non-human donor residue as compared        to the corresponding human germline VH or VL CDR; and    -   (f) for each position within CDR-H3, the residue is any one of        the 20 natural amino acid residues.        E113. The antibody or antigen-binding fragment of embodiment        112, wherein said antibody or antigen-binding fragment binds to        said target antigen with a binding affinity (Kd) value of 5×10⁻⁵        M or less.        E114. The antibody or antigen-binding fragment of embodiment 112        or 113, wherein said antibody or antigen-binding fragment binds        said target antigen with a binding affinity (Kd) value that is        equal or less than the binding affinity (Kd) value of said        non-human donor antibody.        E115. The antibody or antigen-binding fragment of any one of        embodiments 112-114, wherein said human germline VH framework        sequence comprises a VH3 framework sequence.        E116. The antibody or antigen-binding fragment of any one of        embodiments 112-114, wherein said human germline VH framework        sequence comprises a VH1 framework sequence.        E117. The antibody or antigen-binding fragment of any one of        embodiments 112-114, wherein said human germline VH framework        sequence comprises a VH5 framework sequence.        E118. The antibody or antigen-binding fragment of any one of        embodiments 112-114, wherein said human germline VH framework        sequence comprises the VH framework sequence of any one of the        human germline sequences listed in Table 2.        E119. The antibody or antigen-binding fragment of any one of        embodiments 112-114 and 118, wherein said human germline VH        framework sequence comprises the VH framework sequence of human        germline IGHV3-23 or IGHV1-69.        E120. The antibody or antigen-binding fragment of any one of        embodiments 112-114 and 118, wherein said human germline VH        framework sequence comprises the VH framework sequence of human        germline IGHV3-7.        E121. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-114, wherein said human germline VH        framework sequence comprises a VH germ line consensus framework        sequence.        E122. The antibody or antigen-binding fragment thereof of        embodiment 121, wherein said human germline VH framework        sequence comprises the VH framework sequence of any one of the        consensus sequences listed in Table 5.        E123. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122, wherein said human germline VL        framework sequence comprises a V_(K)framework sequence.        E124. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-123, wherein said human germline VL        framework sequence comprises the VL framework sequence of any        one of the human germline sequences listed in Table 3.        E125. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122 wherein said human germline VL        framework sequence comprises a V_(λ) framework sequence.        E126. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122 and 125, wherein said human germline        VL framework sequence comprises the VL framework sequence of any        one of the human germline sequences listed in Table 4.        E127. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122, wherein said human germline VL        framework sequence comprises the VL framework sequence of human        germline IGKV3-20.        E128. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122, wherein said human germline VL        framework sequence comprises the VL framework sequence of human        germline IGKV1-39.        E129. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122, wherein said human germline VL        framework sequence comprises a VL germline consensus framework        sequence.        E130. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-122 and 129, wherein said human germline        VL framework sequence comprises the VL framework sequence of any        one of consensus sequences listed in Table 6.        E131. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-130, wherein said non-human donor        antibody is a non-human mammalian antibody.        E132. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-131, wherein said non-human donor        antibody is a murine antibody, a rat antibody, or a rabbit        antibody.        E133. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-132, wherein said non-human donor        antibody is a murine antibody.        E134. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-133, wherein said human germline VL        framework comprises no more than 3 back-mutations or random        mutations.        E135. The antibody or antigen-binding fragment thereof of any        one of embodiments 131-134, wherein said human germline VH        framework comprises no more than 3 back-mutations or random        mutations.        E136. The antibody or antigen-binding fragment thereof of any        one of embodiments 131-135, wherein said human germline VL        framework and VH framework together comprise no more than 3        back-mutations or random mutations.        E137. The antibody or antigen-binding fragment thereof of any        one of embodiments 131-136, wherein at least one back-mutation        or random mutation is located between heavy chain residues H71        to H80.        E138. The antibody or antigen-binding fragment thereof of any        one of embodiments 131-136, wherein said human germline VH        framework and human germline VH framework do not comprise a        back-mutation.        E139. The antibody or antigen-binding fragment thereof of any        one of embodiments 112-130, wherein said non-human donor        antibody is an avian antibody.        E140. The antibody or antigen-binding fragment thereof of        embodiment 139, wherein said human germline VL framework        comprises no more than 5 back-mutations or random mutations.        E141. The antibody or antigen-binding fragment thereof of        embodiment 139 or 140, wherein said human germline VH framework        comprises no more than 5 back-mutations or random mutations.        E142. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-141, wherein said human germline VL        framework and VH framework together comprise no more than 5        back-mutations or random mutations.        E143. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-142, wherein at least one back-mutation        or random mutation is located between heavy chain residues H71        to H80.        E144. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-143, wherein said human germline VL        framework comprises a single back-mutation.        E145. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-144, wherein said human germline VH        framework comprises a single back-mutation.        E146. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-145, wherein said non-human donor        antibody is a chicken antibody.        E147. The antibody or antigen-binding fragment thereof of any        one of embodiments 139-142 and 144-146, wherein said human        germline VL framework comprises a back-mutation at light chain        position 46 (such as Leu46Thr (L46T)).        E148. The antibody or antigen-binding fragment of any one of        embodiments 112-147, wherein position 24 of CDR-L1 comprises the        corresponding human germline residue from said human VL        germline.        E149. The antibody or antigen-binding fragment of any one of        embodiments 112-148, wherein position 25 of CDR-L1 comprises the        corresponding human germline residue from said human VL        germline.        E150. The antibody or antigen-binding fragment of any one of        embodiments 112-149, wherein position 26 of CDR-L1 comprises the        corresponding human germline residue from said human VL        germline.        E151. The antibody or antigen-binding fragment of any one of        embodiments 112-150, wherein position 27 of CDR-L1 comprises the        corresponding human germline residue from said human VL        germline.        E152. The antibody or antigen-binding fragment of any one of        embodiment 148-151, wherein said human germline VL is a VK        germline.        E153. The antibody or antigen-binding fragment of any one of        embodiments 112-152, wherein position 60 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E154. The antibody or antigen-binding fragment of any one of        embodiments 112-153, wherein position 61 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E155. The antibody or antigen-binding fragment of any one of        embodiments 112-154, wherein position 62 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E156. The antibody or antigen-binding fragment of any one of        embodiments 112-155, wherein position 63 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E157. The antibody or antigen-binding fragment of any one of        embodiments 112-156, wherein position 64 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E158. The antibody or antigen-binding fragment of any one of        embodiments 112-157, wherein position 65 of CDR-H2 comprises the        corresponding human germline residue from said human VH        germline.        E159. The antibody or antigen-binding fragment of any one of        embodiment 153-158, wherein said human germline VH is a VH3        germline.        E160. The antibody or antigen-binding fragment of any one of        embodiment 153-158, wherein said human germline VH is a VH1        germline.        E161. The antibody or antigen-binding fragment of any one of        embodiment 153-158, wherein said human germline VH is a VH5        germline.        E162. The antibody or antigen-binding fragment of any one of        embodiment 153-158, wherein said human germline VH is a VH4        germline.        E163. A nucleic acid molecule encoding the antibody or        antigen-binding fragment thereof of any one of embodiments        112-162.        E164. A host cell comprising the nucleic acid molecule of        embodiment 163.        E165. The host cell of embodiment 164, wherein said host cell is        a CHO cell, a HEK cell, or an Sp2.0 cell.        E166. A method of making an antibody or antigen-binding fragment        thereof, comprising culturing the host cell of embodiment 163 or        164 under a condition wherein said antibody or antigen-binding        fragment is expressed by said host cell.        E167. An antibody or antigen-binding fragment thereof obtained        by the method of any one of embodiments 1-53, 104-111, and 166.        E168. An antibody or antigen-binding fragment thereof obtained        using the library of any one of embodiments 54-100.        E169. A kit comprising the library of any one of embodiments        54-100.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of ABS library design, selection andscreening principles. (A) Amino acid sequences are shown for thev-domains of Par-RAGE and destination germline (DPK9-DP54) scFvs inVL-VH format. At each position where the rat and human residuesdiffered, both residues were encoded for in the ABS-RAGE library (humanresidue on top and rat residue at bottom). This principle was applied toall CDRs other than the CDR-H3, in a single combinatorial library. Inthe CDR-H3, point mutations were permitted at a frequency of 1±1 perclone. (B) Phage libraries were generated and (C) used in selections oncognate antigen. (D) Selection output clones were subsequently screenedby ELISA, HTRF and DNA sequencing to identify hits with maintainedtarget binding and epitope specificity. (E) Top clones were cloned asIgGs, expressed and purified, before (F) characterization of affinity byBiacore, solubility and aggregation analyses by SEC, in vitrospecificity by ELISA and Biacore, and thermal stability analysis by DSC.Human and rat CDR-L1: SEQ ID NOs: 1 and 2, respectively; Human and ratCDR-L2: SEQ ID NOs: 3 and 4, respectively; Human and rat CDR-L3: SEQ IDNOs: 5 and 6, respectively; Human and rat CDR-H1: SEQ ID NOs: 7 and 8,respectively; Human and rat CDR-H2: SEQ ID NOs: 9 and 10, respectively;rat CDR-H3: SEQ ID NO: 11.

FIGS. 2A-2C show the results of biophysical analyses for ABS-derivedclones. DSC analysis of IgG thermal stability for anti-RAGE (A),anti-pTau (B) and anti-A33 (C) antibodies. FIGS. 2D-2F show the resultsof forced IgG aggregation analysis for anti-RAGE (D), anti-pTau (E) andanti-A33 (F) antibodies.

FIGS. 3A-3C show the assessment of potential immunogenicity and level ofhuman identity in the v-domains of ABS-derived leads. (A) EpiMatrix wasused to estimate the potential for T-cell epitope driven immunogenicityin the v-domains of C21-ABS-pTau, C2-ABS-A33 and C7-ABS-RAGE incomparison to 31 FDA-approved therapeutic antibodies that are rodent,humanized or “fully human” in sequence. Lower score suggests lowerpredicted immunogenicity. (B) Comparison of non-human solvent-accessiblesurface area (nhSASA, in Å²) that contributed to the v-domains ofparental (black), grafted (grey) and ABS (white) clones for pTau, A33and RAGE. While CDR grafting dramatically lowers the nhSASA score in allcases, the ABS lead clones all exhibit minimized exposure of non-humanamino acid motifs that might constitute b-cell epitopes. (C) Publicallyavailable, online tools which estimate levels of v-gene sequenceidentity to the human v-domain repertoire were used to calculate T20, Zand G scores for 31 approved therapeutic antibodies that are rodent,humanized or “fully human” in origin, plus the parental, graft(triangles) and ABS leads for pTau (black squares), RAGE (stars) and A33(diamonds).

FIGS. 4A-4B show CDR redundancy analyses in anti-pTau via mutationaltolerance analysis. A plot of chicken amino acid retention frequenciesin the CDRs of the ELISA-positive population of 188 unique clones fromABS-pTau library screening is shown for (A) V_(H) and (B) V_(L) domains,respectively. Only those residues targeted for binary human/chickensubstitution are plotted. CDR residues noted on the X-axis whose valuesare set at 0 were identical human-chicken and not sampled in thelibrary, but are included in the figure for clarity (e.g G26-). In bothplots the mean human-chicken frequency (˜50%) in sequenced clones fromthe starting library is plotted as a dashed line, with standarddeviations as solid bold lines. Residues predicted to be making antigencontacts in a co-crystal structural analysis that were also retainedat >80% (i.e. where mutational tolerance was low) are highlighted withstriped bars. Predicted contact residues not found to be retained arehighlighted in checked bars.

FIGS. 5A-5C show ELISA analyses of scFv function after expression in E.coli from phagemid vector pWRIL-1. Binding signals in titration ELISAagainst: (A) pT231_pS235 peptide for purified V_(L)-V_(H) scFv forms ofPar-pTau and Graft-pTau. (B) hRAGE and mRAGE for periprep V_(L)-V_(H)scFv forms of CL-Hum-pTau and Graft-RAGE. (C) Human A33 ectodomainprotein for purified V_(L)-V_(H) scFv forms of Par-A33 and Graft-A33. Inall cases (A-C), the grafted scFvs show significantly impaired bindingactivity.

FIG. 6 shows sequence alignments for V_(L) and V_(H) domains of 8prioritized lead clones from the ABS-pTau library (from top to bottom,SEQ ID NOs. 12-29). CDR regions are boxed in grey. Amino acids differingfrom the human DPL16/JL2 and DP47/JH4 germlines are underlined. Blackarrow indicates the position of the L46T mutation positively selected inthe FW2 of all clones showing functional binding to target.

FIGS. 7A-7C show the IgG titration HTRF data for representative leadABS-derived clones prioritized on the basis of: % reduction in parentalresidue CDR content and neutralisation of parental IgG binding toantigen, with the change in fluorescence intensity relative to baseline(F/F0) IC₅₀ values approximately equivalent to or better than parentalIgG. (A) Anti-pTau clones, (B) Anti-RAGE clones, (C) Anti-A33 clones(-ve control non-A33-specific IgG added instead of graft due to graftexpression problems).

FIGS. 8A-8D show the results of IgG specificity testing.

FIG. 9 shows in silico predictions for T-cell epitopes within the fullV_(H) and V_(L) regions of lead clones were performed with EpiMatrix(Epivax, RI) (from top to bottom, SEQ ID NOs. 30-49, 45, 50-52, 30,53-55). Possible epitopes are defined as 9-mer sequences with four ormore hits and are shown based on potential epitopes different to humangerm-line, potential epitopes in germ-line sequence to which mostpatients should exhibit self-tolerance, and epitopes in germ-linesequence reported to be potential t-regulatory cell stimulatingsequences. Where potential epitopes overlap the coloring of theC-terminal epitope is used. C7-ABS-RAGE has the lowest immunogenicitypotential of the anti-RAGE clones as it contains 2 potential t-cellepitopes and 13 potential t-regitopes, in comparison to 9 potentialt-cell epitopes and only one t-regitope in Par-RAGE and 2 potentialt-cell epitopes with 10 potential t-regitopes in CL-Hum-RAGE. Notably,the ABS germlining of the c-terminal end of the CDR-H2 (VKD>VKG)reinstates a potential germline t-regitope, as does the germlining ofthe V_(L) FW2. Similarly, C21-ABS-pTau has the lowest immunogenicitypotential of the anti-pTau clones as it contains only 1 potential t-cellepitope and 9 potential t-regitopes, in comparison to 5 potential t-cellepitopes and only one t-regitope in Par-pTau and 2 potential t-cellepitopes with 9 potential t-regitopes in Graft-pTau.

FIGS. 10A-10C are retention frequency plots for all positions across 5CDR-humanized antibodies (A33, pTau, BDNF, IL33, and FN14) using the ABSmethods described herein. FIG. 10A: VH; FIG. 10B: VK; FIG. 10A: Vλ.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

As disclosed herein, conventional antibody humanization “grafts”non-human (e.g., murine) CDRs into a human framework sequence. It isgenerally believed that CDRs are crucial for antigen binding; therefore,the original non-human CDR sequences are maintained, and back-mutationsare introduced into human framework region to restore binding affinityof the grafted CDRs. However, such humanization techniques still retainhigh contents of non-human amino acids. For example, one or more of thenon-human residues in the CDRs may still be immunogenic in human.

Surprisingly, the inventors recognized that the six CDRs can also bemutated in ways that could not be predicted a priori. In fact, due toredundancy of amino acid usage in the antibody paratope, a significantnumber of CDR residues could be substituted, for example, with humangermline residues to further increase the human amino acid content. Forexample, structural analyses illustrated that only subsets of CDRresidues actually make contact with antigen. Remarkably, many CDRresidues do not contact a target antigen directly; instead, these CDRresidues form redundant paratope space that can be used to bind asecond, unrelated antigen (Bostrom et al. Science 1610-4, 2009). Thisfinding challenges the traditional paradigm that describesantibody-antigen interaction as a lock and key (which suggests that eachantibody surface can only accommodate one antigen). If not all CDRresidues are required for binding of a single antigen, then a smallnumber of “redundant” paratope residues may be further mutated withoutsignificantly affecting binding affinities.

Accordingly, as disclosed and exemplified herein, the inventorsdiscovered that “redundant” paratope residues in CDRs can be replacedwith human germline residues to create “ultra” humanized antibodies.

For example, as shown in the examples, the inventors created severalphage display libraries to screen for “ultra” humanized antibodies (withincreased number of human residues in CDRs as compared to theconventional CDR grafting methods). Each library was based on a startingnon-human donor antibody (rat anti-RAGE, rabbit anti-A33, and chickenanti-pTau). As illustrated in FIG. 1A, in each case, five non-humandonor CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2) were alignedwith their corresponding CDRs from a human germline sequence. If a donorresidue is the same as the corresponding human germline residue, thatresidue remained unchanged, and incorporated into all clones of thelibrary. If a donor residue is different from the corresponding humangermline residue, both residues were incorporated combinatorially intolibrary clones.

For example, as shown in FIG. 1, the alignment of the CDR-L1 of ratanti-RAGE antibody and human germline DPK9 is as follows:

TABLE 10 Position 24 25 26 27 28 29 30 31 32 33 34 Human Germline R A SQ S I S S Y L N DPK9 Rat anti-RAGE R A S Q D V G I Y V N antibodyFor positions 24, 25, 26, 27, 32, and 34 (in bold), the human residueand the corresponding rat residue are the same, therefore, all libraryclones incorporate the same residue (e.g., “R” at position 24) at thedesignated position. In certain embodiments, it may be preferable to usehuman germline codon to encode this residue. For positions 28, 29, 30,31, and 33, the human residue and the corresponding rat residue aredifferent. Therefore, only a portion of the library clones comprise thehuman germline residue (e.g., 60% of the library clones comprise thehuman residue “S” at position 28), the remainder of the library clonescomprise the corresponding rat residue (e.g., 40% of the library clonescomprise the rat residue D at position “28”) at the designated position.

In certain embodiments, it may be desirable that for each position,about 50% of the clones have the human germline residue, and about 50%of the clones have the non-human donor residue; so that both residuesare substantially equally represented in the library. However, asdisclosed and exemplified herein, 50%:50% (human:non-human) distributionis not necessary. The libraries of the invention not only toleratesignificant variations in human:non-human distribution, but in somecircumstances, unequal distribution may be desired (e.g., to improvestability).

For CDR-H3, each position can be any one of the 20 natural amino acidresidues. In certain embodiments, it may be desirable that for eachposition, each of the 20 natural amino acid residues are substantiallyequally represented in the library. Again, substantially equaldistribution of the 20 natural amino acid residues is not necessary. Forexample, in some circumstances, the presence of certain residues may bereduced or avoided (e.g., residues that might cause stability problems).

This design principle is named “Augmented Binary Substitution” (ABS).“Binary” means that either human germline residue or non-human donorresidue is used in CDR L1-L3 and H1-H2 to increase the human content ofthe antibody; and “augmented” refers to additional mutations introducedin CDR-H3 to further optimize the activities of the antibody.

Because both human and non-human residues are incorporated into thelibrary clones combinatorially, it is theoretically possible that a verysmall number of library clones have only human germline residues inCDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2 (“all human” clones). That is,each time the human and non-human donor residues differ, the humanresidue is incorporated, resulting in a clone that comprises theoriginal human germline CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2sequences. Conversely, it is also theoretically possible that a verysmall number of library clones have only non-human donor residues inCDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2 (“all donor” clones). That is,each time human and non-human donor residues differ, the non-human donorresidue is incorporated, resulting in a clone that comprises theoriginal non-human donor CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2sequences.

In general, all human and all donor clones should be less than 1%.Assuming that, for each antibody, there are at least seven positions(CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2 combined) where human andnon-human donor residues differ, the number of “all human” libraryclones should be less than 1% (at least 1×2⁷, or 128 individual clones;1 out of 128 is less than 1 percent). Similarly, the number of “allnon-human” library clones should be less than 1% as well. Accordingly,more than 99% of the clones in the library should comprise at least onemore human germline residue in CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2combined as compared to the original donor CDR donor sequences; and morethan 99% of the clones in the library should comprise at least onenon-human donor residue in CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2combined, as compared to the original human germline CDR sequences.

In the CDR-H3, the library may or may not need to be “Augmented” by theaddition of point mutations or random mutations. “Augmentation” mayencourage the “fit” for this loop either into the human v-domains or inbinding to target.

Based on ABS design principle, CDR repertoires (FIG. 1) were built intohuman germline frameworks (DP54 and DPK9 in the Examples), then screenedto identify lead clones with epitope specificity and affinity equivalentto the parental clone. This proved to be a convenient and rapid methodwhich retains the functionally-required CDR residues of the originalnon-human donor antibody, without the need for prior crystal-structureinsight. Notably, this CDR redundancy-minimization approach generatedhighly stable and soluble human IgGs, from multiple key antibodydiscovery species. The resulting antibodies have significantly reducednon-human residue content, such as t-cell epitope content (which is amajor risk factor for antibody immunogenicity in human) and reducednon-human germline surface amino acid exposure, which may lead toreduced B-cell epitope content.

2. Definitions

An antibody “variable domain” refers to the variable region of theantibody light chain (VL) or the variable region of the antibody heavychain (VH), either alone or in combination. As known in the art, thevariable regions of the heavy and light chains each consist of fourframework regions (FR) connected by three complementarity determiningregions (CDRs), and contribute to the formation of the antigen bindingsite of antibodies.

Residues in a variable domain are numbered according Kabat, which is anumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies. See, Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)). Usingthis numbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

Various algorithms for assigning Kabat numbering are available. Thealgorithm implemented in the 2012 release of Abysis (www.abysis.org) isused herein to assign Kabat numbering to variable regions unlessotherwise noted.

The term “Complementarity Determining Regions” (CDRs) are defined asfollows (numbering according to Kabat; H: heavy chain; L: light chain):

-   -   CDR-HI: H26-H35B; CDR-H2: H49-H65; CDR-H3: H95-H102    -   CDR-LI: L24-L34; CDR-L2: L50-L56; CDR-L3: L89-L97        The boundaries of the CDRs defined herein are not identical to        the CDRs originally defined by Kabat (1991), and the definition        of instant disclosure controls.

“Framework” (FR) residues are antibody variable domain residues otherthan the CDR residues. A VH or VL domain framework comprises fourframework sub-regions, FR1, FR2, FR3 and FR4, interspersed with CDRs inthe following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According tothe definition provided herein, FR residues include the following(number according to Kabat; H: heavy chain; L: light chain):

TABLE 11 FR1 FR2 FR3 FR4 Heavy Chain H1-H25 H36-H48 H66-H94 H103-H113Light Chain L1-L23 L35-L49 L57-L88 L98-L107

An “antigen-binding fragment” of an antibody refers to a fragment of afull-length antibody that retains the ability to specifically bind to anantigen (preferably with substantially the same binding affinity).Examples of an antigen-binding fragment includes (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR),disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies andintrabodies. Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv)); see e.g., Birdet. al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988)). Other forms of single chain antibodies,such as diabodies are also encompassed. Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see e.g., Holliger et al. Proc.Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure2:1121-1123).

A “human antibody” is an antibody comprising an amino acid sequence thatis derived from a human germline, such as an antibody expressed by ahuman B cell, or an antibody expressed by a transgenic animal thatcomprises a nucleic acid sequence encoding a human immunoglobulin. Alsoincluded herein is an antibody that comprises a consensus human antibodysequence. For example, the framework sequence of a human antibody can bea specific human germline framework sequence (e.g., Tables 2-4), or aconsensus human germline framework sequence (e.g., Tables 5-6). Thisdefinition of a human antibody specifically excludes a humanizedantibody comprising non-human CDR sequences.

A “non-human donor antibody” includes any antibody that is not a “humanantibody” defined herein. A non-human donor antibody can be an antibodycomprising an amino acid sequence that corresponds to an immunoglobulinproduced by non-human species. The CDR residues (and a selected numberof framework residues) of a non-human antibody can be used as “donor”residues during humanization process. Also included herein is aCDR-grafted antibody in which CDR sequences from a non-human species(such as murine) are grafted into a human framework. One aspect of theinvention is to further humanize CDR-grafted, humanized antibody, byintroducing additional human germline residues in the CDR region.Therefore, a CDR-grafted, humanized antibody can also serve as a“non-human donor” antibody.

“Corresponding” CDR or FR residues from different antibodies can beidentified according to sequence alignment or structural alignment thatis known in the art. For example, “corresponding” CDR or FR residuesfrom different antibodies can be identified by alignment according toKabat numbering, or any other numbering systems that are known in theart, such as AHo, IMGT, Chothia, or AbM. “Corresponding” CDR or FRresidues share the same numbers under such a numbering system.Alternatively, alignments can be done by hand or by using well-knownsequence alignment programs such as ClustalW2, or “BLAST 2 Sequences”using default parameters. For example, NCBI “IgBLAST” is specificallyfor antibodies.

In addition to sequence alignment, structural alignment may also be usedto identify “corresponding” CDR or FR residues. Structural alignmentsuse information about the secondary and tertiary structure to aid inaligning the sequences. These methods are used for two or more sequencesand typically produce local alignments; however, because they depend onthe availability of structural information, they can only be used forsequences whose corresponding structures are known (usually throughX-ray crystallography or NMR spectroscopy). Sometimes, structuralalignments can be more reliable between sequences that are verydistantly related and that have diverged so extensively that sequencecomparison cannot reliably detect their similarity. Where there is noavailable structural data on one of the proteins, a comparison can stillbe made if structural data is available on one or preferably moreclosely related proteins, such as immunoglobulins across species, and inparticular antibody constant domains across species and subtype. Acommonly used algorithm for structural alignments is TM-ALIGN (Zhang andSkolnick, Nucleic Acids Research, 33: 2302-2309 (2005)), which assignsincreased weight to the most similar regions of the structure duringsuperposition.

In certain embodiments, one or more “back-mutations” may be used duringCDR grafting. A back-mutation refers to a mutation in antibody variabledomain framework region where a human germline residue is replaced withthe corresponding non-human donor residue to increase the antigenbinding affinity of a humanized antibody. A “random mutation” refers tothe substitution of an amino acid residue with a different amino acidresidue.

Specific amino acid residue positions are also numbered according toKabat. For example, for human VK1 germline IGKV1-39 used in theexamples, “Leu46” (or L46) refers to position 46 according to Kabatnumbering (which is a Leu). However, the “Leu46” (or L46) designationincludes any residue from another antibody (e.g., an antibody fromanother human or non-human antibody) that corresponds to Leu46 of humanVK1 germline IGKV1-39 (even though the actual position of that residuemay or may not be 46, and the actual residue may or may not be Leu). Forexample, for human VK1D germline IGKV1D-16, position 46 (Kabatnumbering) is Ser, and VK1D germline IGKV1 D-17, position 46 (Kabatnumbering) is His. Therefore, for simplicity, Leu46 (or L46) is used torefer a residue that aligns with Leu46 of IGKV1-39, even if it is a Seror His. Similarly, mutations are also identified according to Kabatnumbering. For example, Leu46Thr (or “L46T”) means that a residue froman antibody that corresponds to Leu46 of human germline IGKV1-39 (whichmay or may not be Leu) is mutated to Thr.

The binding affinity of an antibody can be expressed as Kd value, whichrefers to the dissociation rate of a particular antigen-antibodyinteraction. Kd is the ratio of the rate of dissociation, also calledthe “off-rate (koff)”, to the association rate, or “on-rate (kon)”.Thus, Kd equals koff/kon and is expressed as a molar concentration (M),and the smaller the Kd, the stronger the affinity of binding. Kd valuesfor antibodies can be determined using methods well established in theart. One exemplary method for measuring Kd is surface plasmon resonance(SPR), typically using a biosensor system such as a BIACORE® system.BIAcore kinetic analysis comprises analyzing the binding anddissociation of an antigen from chips with immobilized molecules (e.g.molecules comprising epitope binding domains), on their surface. Anothermethod for determining the Kd of an antibody is by using Bio-LayerInterferometry, typically using OCTET® technology (Octet QKe system,ForteBio). For example, a standard assay condition for surface plasmonresonance can be based on ligand immobilization of approximately 100Response Units (RU) of IgG on the SPR chip. Purified target proteins arediluted in buffer to a range of final concentrations and injected at arequisite flow rate (e.g. 10-100 μl/min) to allow the calculation ofK_(a). Dissociation is allowed to proceed to establish off-rate (K_(d)),followed by a 5 sec pulse of 20 mM NaOH for regeneration of the chipsurface. Sensorgrams are then analyzed using a kinetics evaluationsoftware package.

The term “about”, as used here, refers to +/−10% of a value.

3. Humanized Antibodies and Antibody Libraries

Provided herein are libraries for humanizing a non-human donor antibodythat binds to a target antigen, wherein: (a) said library comprises aplurality of polypeptides, each polypeptide comprising an antibodyvariable domain; (b) said antibody variable domain comprises (i) a VHdomain comprising: a human germline VH framework sequence, and CDR-H1,CDR-H2, and CDR-H3; and (ii) a VL domain comprising: a human germline VLframework sequence, and CDR-L1, CDR-L2, and CDR-L3; (c) for eachindividual position within said CDR-L1, CDR-L2, CDR-L3, CDR-H1, andCDR-H2: if the human germline residue at said position is the same asthe corresponding non-human donor residue, all polypeptides in thelibrary comprise the human germline residue at said position; if thehuman germline residue at the position is different from thecorresponding non-human donor residue, a portion of the polypeptides inthe library comprise the human germline residue at said position, theremainder of the polypeptides comprise the corresponding non-human donorresidue at said position; (d) for each individual position withinCDR-H3, the residue is any one of the 20 natural amino acid residues. Inaddition, less than 1% of the polypeptides in said library comprise theoriginal non-human donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2sequences; and less than 1% of the polypeptides in said library comprisethe original human VL germline CDR-L1, CDR-L2, and CDR-L3, and theoriginal human VH germline CDR-H1 and CDR-H2 sequences.

The libraries disclosed herein can be used to screen for “ultra”humanized antibodies, in particular antibodies where human germlineresidues are incorporated into non-human donor CDRs. Accordingly, alsoprovided herein is humanized antibody or antigen-binding fragmentthereof that binds to a target antigen, wherein: (a) said antibody orantigen-binding fragment thereof comprises (i) a VH domain comprising: ahuman germline VH framework sequence, and CDR-H1, CDR-H2, and CDR-H3;and (ii) a VL domain comprising: a human germline VL framework sequence,and CDR-L1, CDR-L2, and CDR-L3; (b) said CDR-L1, CDR-L2, CDR-L3, CDR-H1,and CDR-H2 are derived from corresponding CDRs from a non-human donorantibody that binds to said target antigen; (c) for each position withinsaid CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, the residue is eitherhuman germline residue from said human germline VL or VH, orcorresponding residue from said non-human donor antibody; (d) saidCDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each comprises at least onemore human germline residue as compared to the corresponding non-humandonor CDR, (e) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 eachcomprises at least one more non-human donor residue as compared to thecorresponding human germline VH or VL CDR; and (f) for each positionwithin CDR-H3, the residue is any one of the 20 natural amino acidresidues.

A. Non-human Donor Antibodies

Humanization generally starts with obtaining CDR sequences from anon-human donor antibody that binds to a target antigen, andincorporating non-human donor residues into a human framework.

Non-human donor antibody binds to a target antigen and can be obtained,e.g., by conventional techniques (such as hybridoma technology,recombinant DNA technology). For example, the target antigen may beisolated from a natural source, or may be produced recombinantly or byin vitro synthesis. Alternatively, cells comprising native orrecombinant antigen can be used. The antigen can be administered to asuitable non-human host to induce production of antibodies. Monoclonalantibodies can then be obtained by, for example, hybridoma technology.

Multiple non-human donor antibodies can be screened to select anantibody that has strong binding affinity for the target antigen. Forexample, the non-human donor antibody may bind the antigen of interestwith a binding affinity (Kd) value of about 1×10⁻⁵ M or less, such asabout 1×10⁻⁵ M or less, about 1×10⁻⁶ M or less, about 1×10⁻⁷ M or less,about 1×10⁻⁸ M or less, about 1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less,about 1×10⁻¹ M or less, about 1×10⁻¹² M or less, about 1×10⁻¹³ M orless, from about 1×10⁻⁵ M to about 1×10⁻¹³ M, from about 1×10⁻⁶ M toabout 1×10⁻¹³ M, from about 1×10⁻⁷ M to about 1×10⁻¹³ M, from about1×10⁻⁸ M to about 1×10⁻¹³ M, from about 1×10⁻⁹ M to about 1×10⁻¹³ M,from about 1×10⁻⁵ M to about 1×10⁻¹² M, from about 1×10⁻⁶ M to about1×10⁻¹² M, from about 1×10⁻⁷ M to about 1×10⁻¹² M, from about 1×10⁻⁸ Mto about 1×10⁻¹² M, from about 1×10⁻⁹ M to about 1×10⁻¹² M, from about1×10⁻⁵ M to about 1×10¹¹ M, from about 1×10⁻⁶ M to about 1×10¹¹ M, fromabout 1×10⁻⁷ M to about 1×10¹¹ M, from about 1×10⁻⁸ M to about 1×10¹¹ M,or from about 1×10⁻⁹ M to about 1×10¹¹ M. Generally, the antibody willbind antigen with an affinity in the nanomolar or better range.

The sequence of the non-human donor antibody can be determined usingstandard sequencing techniques, or obtained from a sequence database orother literature resources. If desired, polynucleotide sequence(s)encoding the antibody may then be cloned into a vector for expression orpropagation.

The CDR and framework sequences of the non-human donor antibody can bereadily ascertained using standard antibody numbering systems, such asKabat numbering.

Examples of antigens include: HER2, CD20, TNF ALPHA, C5, C5a, CD30,Blys, CTLA-4, IL1B, PD-1, PDL-1, IL12, IL23, IL17a, VEGF, EGFR, IL-6R,CD11a, APLHA-4-INTEGRIN, IgE, CD52, CD33, CD25, RSV, B. anthracisprotective antigen, CD3, IL33, P-CADHERIN, NOTCH1, EPHA4, 5T4, IL4,IL13, MADCAM, IL6, 41BB, OX-40, TFPI, CXCR4, and FGF21.

Additional examples of antigens include: PDGFRα, PDGFRβ, PDGF, VEGF,VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGFR1, VEGFR2, VEGFR3,FGF, FGF2, HGF, KDR, fit-1, FLK-1, Ang-2, Ang-1, PLGF, CEA, CXCL13,Baff, IL-21, CCL21, TNF-α, CXCL12, SDF-I, bFGF, MAC-I, IL23pl9, FPR,IGFBP4, CXCR3, TLR4, CXCR2, EphA2, EphA4, EphrinB2, EGFR(ErbB1),HER2(ErbB2 or p185neu), HER3(ErbB3), HER4 ErbB4 or tyro2), SCI, LRP5,LRP6, RAGE, s100A8, s100A9, Navl.7, GLPI, RSV, RSV F protein, InfluenzaHA protein, Influenza NA protein, HMGBI, CD16, CD19, CD20, CD21, CD28,CD32, CD32b, CD64, CD79, CD22, ICAM-I, FGFRI, FGFR2, HDGF, EphB4, GITR,β-amyloid, hMPV, PIV-I, PIV-2, OX40L, IGFBP3, cMet, PD-I, PLGF,Neprolysin, CTD, IL-18, IL-6, CXCL-13, IL-IRI, IL-15, IL-4R, IgE, PAI-I,NGF, EphA2, uPARt, DLL-4, αvβ5, αvβ6, α5β1, α3β1, interferon receptortype I and type II, CD 19, ICOS, IL-17, Factor II, Hsp90, IGF, IGF-I,IGF-II, CD 19, GM-CSFR, PIV-3, CMV, IL-13, IL-9, and EBV.

Additional examples of antigens include: Tumor Necrosis Factor-α(“TNF-α”), Tumor Necrosis Factor-β (“TNF-β”), Lymphotoxin-α (“LT-α”),CD30 ligand, CD27 ligand, CD40 ligand, 4-1 BB ligand, Apo-1 ligand (alsoreferred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referredto as TRAIL), Apo-3 ligand (also referred to as TWEAK), osteoprotegerin(OPG), APRIL, RANK ligand (also referred to as TRANCE), TALL-I (alsoreferred to as BlyS, BAFF or THANK), DR4, DR5 (also known as Apo-2,TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2, or KILLER), DR6, DcRI, DcR2,DcR3 (also known as TR6 or M68), CARI, HVEM (also known as ATAR or TR2),GITR, ZTNFR-5, NTR-I, TNFLI, CD30, LTBr, 4-1BB receptor and TR9.

Additional examples of antigens include: 5T4, ABL, ABCB5, ABCFI, ACVRI,ACVRIB, ACVR2, ACVR2B, ACVRLI, ADORA2A, Aggrecan, AGR2, AICDA, AIFI,AIGI, AKAPI, AKAP2, AMH, AMHR2, angiogenin (ANG), ANGPTI, ANGPT2,ANGPTL3, ANGPTL4, Annexin A2, ANPEP, APC, APOCI, AR, aromatase, ATX,AXI, AZGPI (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAGI,BAII, BCR, BCL2, BCL6, BDNF, BLNK, BLRI (MDR15), BlyS, BMP1, BMP2, BMP3B(GDFIO), BMP4, BMP6, BMP7, BMP8, BMP9, BMP11, BMP12, BMPR1A, BMPR1B,BMPR2, BPAGI (plectin), BRCAI, C19orflO (IL27w), C3, C4A, C5, C5R1,CANTI, CASPI, CASP4, CAVI, CCBP2 (D6/JAB61), CCLI (1-309), CCLI 1(eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC),CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21(MEP-2), SLC, exodus-2, CCL22(MDC/STC-I), CCL23 (MPIF-I), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26(eotaxin-3), CCL27 (CTACK/ILC),CCL28, CCL3 (MIP-Ia), CCL4 (MIP-Ib), CCL5(RANTES), CCL7 (MCP-3), CCL8(mcp-2), CCNAI, CCNA2, CCNDI, CCNEI, CCNE2, CCRI (CKRI/HM145), CCR2(mcp-IRB/RA),CCR3 (CKR3/CMKBR3), CCR4, CCR5(CMKBR5/ChemR13), CCR6(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1),CCR8 (CMKBR8/TERI/CKR-LI),CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR),CD164, CD19, CDIC, CD20,CD200, CD-22, CD24, CD28, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z,CD4, CD40, CD40L, CD44, CD45RB, CD46, CD52, CD69, CD72, CD74, CD79A,CD79B, CD8, CD80, CD81, CD83, CD86, CD105, CD137, CDHI (E-cadherin),CDCP1CDH10, CDH12, CDH13, CDH18,CDH19, CDH20, CDH5, CDH7, CDH8, CDH9,CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKNIA (p21WapI/CipI), CDKNIB(p27KipI), CDKNIC, CDKN2A (pl61INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB,CERI, CHGA, CHGB, Chitinase, CHSTIO, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5,CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-7), CLN3, CLU(clusterin), CMKLRI, CMKORI (RDCI), CNRI, COLI 8AI, COL1A1.COL4A3,COL6A1, CR2, Cripto, CRP, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF),CTLA4, CTL8, CTNNBI (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYDI),CX3CR1 (V28), CXCLI(GROI), CXCLIO (IP-IO), CXCLII (I-TAC/IP-9), CXCL12(SDFI), CXCL13, CXCL 14,CXCL 16, CXCL2 (GR02), CXCL3 (GR03), CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9/CKR-L2), CXCR4,CXCR6 (TYMSTR/STRL33/Bonzo), CYB5, CYCI, Cyr61, CYSLTRI, c-Met, DAB21P,DES, DKFZp451J0118, DNCLI, DPP4, E2F1, ECGFI5EDGI, EFNAI, EFNA3, EFNB2,EGF, ELAC2, ENG, endoglin, ENOI, EN02, EN03, EPHAI, EPHA2, EPHA3, EPHA4,EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHAIO, EPHBI, EPHB2, EPHB3, EPHB4,EPHB5, EPHB6, EPHRIN-AI, EPHRIN-A2, EPHRIN-A3, EPHRIN-A4, EPHRIN-A5,EPHRIN-A6, EPHRIN-BI, EPHRIN-B2, EPHRTN-B3, EPHB4,EPG, ERBB2 (Her-2),EREG, ERK8, Estrogen receptor, ESRI, ESR2, F3 (TF), FADD,farnesyltransferase, FasL, FASNf, FCER1A,FCER2, FCGR3A, FGF, FGFI(aFGF), FGFIO, FGFI 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18,FGF19, FGF2 (bFGF), FGF20, FGF21 (such as mimAb1), FGF22, FGF23, FGF3(int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR3,FIGF (VEGFD), FILI(EPSILON), FBLI (ZETA), FLJ12584, FLJ25530, FLRTI(fibronectin), FLTI, FLT-3, FOS, FOSLI(FRA-I), FY (DARC), GABRP (GABAa),GAGEBI, GAGECI, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GDF8, GFII, GGTI,GM-CSF, GNASI, GNRHI, GPR2 (CCRIO), GPR31, GPR44, GPR81 (FKSG80), GRCCIO(CIO), gremlin, GRP, GSN (Gelsolin), GSTPI, HAVCR2, HDAC, HDAC4, HDACS5,HDAC7A, HDAC9, Hedgehog, HGF, HIFIA, HIPI, histamine and histaminereceptors, HLA-A, HLA-DRA, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL,ID2, IFN-α, IFNAI, IFNA2, IFNA4,1FNA5, EFNA6, BFNA7, IFNBI, IFNgamma,IFNWI, IGBPI, IGFI, IGFIR, IGF2, IGFBP2,IGFBP3, IGFBP6, DL-I, ILIO,ILIORA, ILIORB, IL-1, ILIRI (CD121a), ILIR2(CD121b), IL-IRA, IL-2, IL2RA(CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5,IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA (CD126), IR6RB(CD130), IL-7,IL7RA(CD127), IL-8, CXCRI (IL8RA), CXCR2 (IL8RB/CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL1IRA, IL-12,IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15,IL15RA, 1L16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1,IL18RAP, IL19, ILIA, ILIB, ILIFIO, IL1F5, IL1F6, IL1F7, IL1F8, DL1F9,ILIHYI, ILIRI, IL1R2, ILIRAP, ILIRAPLI, IL1 RAPL2, ILIRLI, IL1RL2,ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23,DL24, IL25,IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA,IL4,IL4R, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4,IRAKI, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α 6 integrin), ITGAV, ITGB3,ITGB4 (β 4 integrin), JAKI, JAK3, JTB, JUN, K6HF, KAII, KDR, KIM-1,KITLG, KLF5 (GC Box BP), KLF6, KLKIO, KLK12, KLK13, KLK14, KLK15, KLK3,KLK4, KLK5, KLK6, KLK9, KRTI, KRT19 (Keratin 19), KRT2A, KRTHB6(hair-specific type II keratin), LAMA5, LEP (leptin), Lingo-p75,Lingo-Troy, LPS, LRP5, LRP6, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2,LTBR, MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MCP-I, MDK, MIBI, midkine,MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3(metallothionectin-Ui), mTOR, MTSSI, MUCI (mucin), MYC, MYD88, NCK2,neurocan, neuregulin-1, neuropilin-1, NFKBI, NFKB2, NGFB (NGF), NGFR,NgR-Lingo, NgR-Nogo66 (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH,NOTCHI, N0X5, NPPB, NROBI, NR0B2, NRIDI, NR1D2, NR1H2, NR1H3, NR1H4,NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1,NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRPI, NRP2, NT5E, NTN4,OCT-1, ODZ1, OPN1, OPN2, OPRDI, P2RX7, PAP, PARTI, PATE, PAWR, PCA3,PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, peg-asparaginase, PF4(CXCL4), Plexin B2 (PLXNB2), PGF, PGR, phosphacan, PIAS2, PI3 Kinase,PIK3CG, PLAU (uPA), PLG5PLXDCI, PKC, PKC-β, PPBP (CXCL7), PPID, PRI,PRKCQ, PRKDI, PRL, PROC, PROK2, pro-NGF, prosaposin, PSAP, PSCA, PTAFR,PTEN, PTGS2 (COX-2), PTN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGSI,RGS13, RGS3, RNFI10 (ZNF144), Ron, ROB02, RXR, selectin, S100A2, S100A8,S100A9, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2),SCGB2A2(mammaglobin 1), SCYEI (endothelial Monocyte-activating cytokine), SDF2,SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PAI-I), SERPINFI,SHIP-I, SHIP-2, SHBI, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1,SLIT2, SPPI, SPRRIB (SprI), ST6GAL1, STABI, STAT6, STEAP, STEAP2,SULF-1, Sulf-2, TB4R2, TBX21, TCPIO, TDGFI, TEK, TGFA, TGFBI, TGFBIII,TGFB2,TGFB3, TGFBI, TGFBRI, TGFBR2, TGFBR3, THIL, THBSI(thrombospondin-1), THBS2/THBS4, THPO, TIE (Tie-1), TIMP3, tissuefactor, TIKI2, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6JLR7, TLR8, TLR9,TM4SF1, TNF, TNF-α, TNFAIP2 (B94), TNFAIP3, TNFRSFIIA, TNFRSFIA,TNFRSFIB, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9,TNFSFIO (TRAIL), TNFSFI 1 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April),TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF 18, TNFSF4 (OX40ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand),TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-likereceptors, TLR2, TLR4, TLR9, TOP2A (topoisomerase lia), TP53, TPMI,TPM2,TRADD, TRAFI, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREMI,TREM2, TRPC6, TROY, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC,versican, VHL C5, VLA-4, Wnt-1, XCLI (lymphotactin), XCL2 (SCM-Ib), XCRI(GPR5/CCXCRI), YYI, and ZFPM2.

Examples of non-human donor antibodies can be a non-human mammalianantibody (e.g., murine antibody, a rat antibody, a rabbit antibody, allama antibody, an alpaca antibody), or an avian antibody (e.g., achicken antibody or from any domesticated or non-domesticated bird). Acamelid VHH single domain antibody (llama, alpaca or dromedary) may alsobe used. A CDR-grafted, humanized antibodies can also be used as adonor, to further humanize such antibodies by introducing additionalhuman germline residues in the CDR region.

B. Human Frameworks

Sequences of human germline frameworks are available from various publicdatabases, such as V-base, IMGT, NCBI, or Abysis. Exemplary humanframework sequences are listed in Tables 2-6.

Suitable human framework can be the framework region from a particularhuman germline (Tables 2-4), or can be framework region of consensusgermline sequences (Tables 5, 6).

Preferred human germline heavy chain frameworks are frameworks derivedfrom VH1, VH3, or VH5 germlines. For example, VH frameworks from thefollowing germlines may be used: IGHV3-23, IGHV3-7, or IGHV1-69(germline names are based on IMGT germline definition).

Preferred human germline light chain frameworks are frameworks derivedfrom V_(K) or V_(λ) germlines. For example, VL frameworks from thefollowing germlines may be used: IGKV1-39 or IGKV3-20 (germline namesare based on IMGT germline definition).

One exemplary method of selecting a suitable human framework is basedsequence homology between non-human donor framework sequence and humanframework sequences. For example, one can align the non-human donorframework sequence with various human framework sequences, and selectthe most homologous framework sequence. Alternatively, one may alsoselect a framework on the basis of structural complimentarity (e.g.,similarity in canonical CDR structure and therefore CDR presentationcomplimentarity).

As exemplified herein, in many cases, back-mutations in framework region(where a human germline residue is replaced with the correspondingnon-human donor residue to restore binding affinity) is not required forthe ABS method. For example, when the donor CDRs are from a mammalianspecies, high affinity humanized antibody were obtained withoutframework back-mutations (see Examples section). When the donor CDRs arefrom an avian species, only one back-mutation was made (see Examplessection).

Accordingly, when the donor CDRs are from a mammalian species, incertain embodiment, the human germline VL framework comprises no morethan 5 back-mutations or random mutations (such as 5, 4, 3, 2, or 1back-mutation or random mutation); in certain embodiments, the humangermline VH framework comprises no more than 5 back-mutations or randommutations (such as 5, 4, 3, 2, or 1 back-mutations or random mutation);in certain embodiments, the human germline VL framework and VH frameworktogether comprise no more than 5 back-mutations or random mutations(such as 5, 4, 3, 2, or 1 back-mutation or random mutation); in certainembodiments, the human germline VL framework and VH framework togetherdoes not comprise a back-mutation or random mutation.

In particular, 1 or more back-mutations or random mutations can occur inheavy chain FR3, at positions H71-H80. Structural studies show thatresidues H71-H80 form a loop and play an auxiliary role for antigenbinding in many natural antibodies. Some even refer to this region as“CDR4.” The structure-based database indicates that this region canaccommodate diversities comparable to those observed in the classicalCDRs. Further, there appears to be no significant structural constrainton the diversity within the central portion of the loop. Accordingly, ifdesired, positions H71-H80 (in FR3) of heavy chain can be furthermutated. In certain embodiment, positions H71-H80 (in FR3) of heavychain are constructed in binary substitution form—each position is humangermline residue or corresponding non-human donor residue, in a fashionsimilar to that of CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2. Incertain embodiment, positions H71-H80 (in FR3) of heavy chain arerandomized—each position within H71-H80 can be any one of the 20 naturalamino acids, in a fashion similar to that of CDR-H3.

When the donor CDRs are from an avian species, in certain embodiment,the human germline VL framework comprises no more than 10 back-mutationsor random mutations (such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1back-mutation or random mutation); in certain embodiments, the humangermline VH framework comprises no more than 10 back-mutations or randommutations (such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 back-mutation orrandom mutation); in certain embodiments, the human germline VLframework and VH framework together comprise no more than 10back-mutations or random mutations (such as 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 back-mutation or random); in certain embodiments, the humangermline VL framework and VH framework together comprise a singleback-mutation or random mutation.

In an exemplary embodiment, the human germline VL framework comprises aback-mutation at position 46.

In an exemplary embodiment, the non-human CDRs are from a chickenantibody, and the back-mutation is at Leu46Thr (L46T).

As described above, 1 or more back-mutations or random mutations canoccur in heavy chain FR3, at positions H71-H80.

C. Humanization of Donor CDRs

The ABS method disclosed herein modifies the donor CDR residues toincrease the human content of donor CDRs.

As illustrated in FIG. 1A, five non-human donor CDRs (CDR-L1, CDR-L2,CDR-L3, CDR-H1, and CDR-H2) are aligned with their corresponding CDRsfrom a human germline sequence. If a donor residue is the same as thecorresponding human germline residue, that residue remained unchanged,and all library clones incorporate this residue at the designatedposition. If a donor residue is different from the corresponding humangermline residue, both residues are incorporated combinatorially intolibrary clones (i.e., a portion of the polypeptides in the librarycomprise the human germline residue at the designated position, theremainder of the polypeptides comprise the corresponding non-human donorresidue at the designated position.

In certain embodiments, it may be desirable that for each position,about 50% of the clones had the human germline residue, and about 50% ofthe clones have the non-human donor residue; so that both residues aresubstantially equally represented in the library. However, it should beunderstood that even if the synthesis scheme is carried out to achievethe goal of 50%/50% for human/non-human distribution, certain synthesisbiases may exist, and substantially equal distribution may not beachieved. For example, experimental and/or mechanical error in thesynthesis methods used to generate DNA libraries may lead to theimprecise incorporation of individual nucleotides or codons such that50:50 distribution of human to non-human is not achieved. In fact, inmany cases, substantially equal distribution of human/non-human residuesis also not necessary.

Accordingly, in certain embodiments, for each position within CDR-L1,CDR-L2, CDR-L3, CDR-H1, and CDR-H2, the percentage of polypeptides inthe library comprising the human germline residue can be from about 1%to about 99%, such as from about 5% to about 95%, from about 10% toabout 95%, from about 15% to about 95%, from about 20% to about 95%,from about 25% to about 95%, from about 30% to about 95%, from about 35%to about 95%, from about 40% to about 95%, from about 5% to about 90%,from about 10% to about 90%, from about 15% to about 90%, from about 20%to about 90%, from about 25% to about 90%, from about 30% to about 90%,from about 35% to about 90%, from about 40% to about 90%, from about 5%to about 85%, from about 10% to about 85%, from about 15% to about 85%,from about 20% to about 85%, from about 25% to about 85%, from about 30%to about 85%, from about 35% to about 85%, from about 40% to about 85%,from about 5% to about 80%, from about 10% to about 80%, from about 15%to about 80%, from about 20% to about 80%, from about 25% to about 80%,from about 30% to about 80%, from about 35% to about 80%, from about 40%to about 80%, from about 5% to about 75%, from about 10% to about 75%,from about 15% to about 75%, from about 20% to about 75%, from about 25%to about 75%, from about 30% to about 75%, from about 35% to about 75%,from about 40% to about 75%, from about 5% to about 70%, from about 10%to about 70%, from about 15% to about 70%, from about 20% to about 70%,from about 25% to about 70%, from about 30% to about 70%, from about 35%to about 70%, from about 40% to about 70%, from about 5% to about 65%,from about 10% to about 65%, from about 15% to about 65%, from about 20%to about 65%, from about 25% to about 65%, from about 30% to about 65%,from about 35% to about 65%, from about 40% to about 65%, from about 5%to about 60%, from about 10% to about 60%, from about 15% to about 60%,from about 20% to about 60%, from about 25% to about 60%, from about 30%to about 60%, from about 35% to about 60%, from about 40% to about 60%,or about 50%, the remainder comprising the corresponding non-human donorresidue at the designated position.

In certain embodiments, it may be preferable to reduce the content ofcertain amino acids that impart chemical instability or heterogeneityproblems, such as methionine, aspartic acid, tryptophan, asparagine,cysteine, tryptophan. Often, these residues are involved inpost-transcriptional modifications, such as glycosylation, methylation,acetylation, oxidation, acid hydrolysis and deamination. Certain typesof post-transcriptional modifications may be undesirable.

For example, as shown in FIG. 1, the human residue at position 53 ofCDR-L2 is S, and the rat residue is N. If desired, the percentage oflibrary clones incorporating N may be reduced to significantly below 50%to minimize the risk of generating daughter clones with deamidation orglycosylation motifs. For example, synthesis may be carried out suchthat only about 1-2% of the clones have N, and 98-99% of the clones haveS. Other circumstances where one may not wish to achieve a 50:50distribution of human: non-human amino acids at given positions could beto avoid the generation of structural motifs that affect protein drugdevelopment such as surface charge patches, and/or hydrophobicitypatches.

For CDR-H3, each position can be any one of the 20 natural amino acidresidues. In certain embodiments, it may be desirable that for eachposition, each of the 20 natural amino acid residues are substantiallyequally represented in the library (i.e., about 5% the library clonesincorporate one particular amino acid). Again, because synthesis biases,substantially equal distribution of the 20 residues may not be achieve,and in many cases, is not necessary. Accordingly, in certainembodiments, for each position within CDR-H3, each of the 20 amino acidresidues is represented by at least about 0.1% (e.g., at least about0.1%, at least about 0.2%, at least about 0.5%, at least about 0.8%, atleast about 1%, at least about 1.5%, at least about 2%, at least about2.5%, at least about 3%, at least about 3.5%, at least about 4%, or atleast about 4.5%) of the polypeptides in the library. In certainembodiments, for each position within CDR-H3, each of the 20 amino acidresidues is represented by from about 0.1% to about 20% (e.g., fromabout 0.1% to about 20%, from about 0.1% to about 15%, from about 0.1%to about 10%, from about 0.2% to about 20%, from about 0.2% to about15%, from about 0.2% to about 10%, from about 0.5% to about 20%, fromabout 0.5% to about 15%, from about 0.5% to about 10%, from about 0.8%to about 20%, from about 0.8% to about 15%, from about 0.8% to about10%, from about 1% to about 20%, from about 1% to about 15%, from about1% to about 10%, from about 2% to about 20%, from about 2% to about 15%,from about 2% to about 10%, from about 3% to about 20%, from about 3% toabout 15%, from about 3% to about 10%, from about 4% to about 20%, fromabout 4% to about 15%, from about 4% to about 10%, from about 0.1% toabout 9%, from about 0.2% to about 9%, from about 0.5% to about 9%, fromabout 0.8% to about 9%, from about 1% to about 9%, from about 2% toabout 9%, from about 3% to about 9%, from about 4% to about 9%, fromabout 0.1% to about 8%, from about 0.2% to about 8%, from about 0.5% toabout 8%, from about 0.8% to about 8%,from about 1% to about 8%, fromabout 2% to about 8%, from about 3% to about 8%, from about 4% to about8%, from about 0.1% to about 7%, from about 0.2% to about 7%, from about0.5% to about 7%, from about 0.8% to about 7%, from about 1% to about7%, from about 2% to about 7%, from about 3% to about 7%, from about 4%to about 7%, from about 0.1% to about 6%, from about 0.2% to about 6%,from about 0.5% to about 6%, from about 0.8% to about 6%, from about0.1% to about 6%, from about 0.2% to about 6%, from about 0.5% to about6%, from about 0.8% to about 6%, from about 1% to about 6%, from about2% to about 6%, from about 3% to about 6%, or from about 4% to about 6%)of the polypeptides in the library.

In certain embodiments, it may be preferable to reduce the content ofcertain amino acids in CDR-H3 that impart chemical instability orheterogeneity problems such as methionine, aspartic acid, tryptophan,asparagine, cysteine, tryptophan, as described above.

Often it is not necessary for the library to incorporate all 20 naturalamino acids at each position within CDR-H3. Since conservativesubstitutions are generally well tolerated, certain residues may beomitted from the library. For example, since Gin is a conservativesubstitution of Asn, the library can omit Asn in CDR-H3, and use Gininstead. Sometimes it may be desirable to increase the percentage of Ginif Asn is replaced with Gin in CDR-H3. Other conservative substitutionscan be similarly used to omit certain residues.

TABLE 12 Conservative Conservative Residue substitution Residuesubstitution Ala Ser Leu Ile, Val Arg Lys Lys Arg, Gln Asn Gln; His MetLeu, Ile Asp Glu Phe Met, Leu, Tyr Cys Ser Ser Thr; Gly Gln Asn Thr Ser,Val Glu Asp Trp Tyr Gly Pro Tyr Trp, Phe His Asn, Gln Val Ile, Leu IleLeu, Val Pro —

Methods of incorporating both human and non-human residues (CDR-L1,CDR-L2, CDR-L3, CDR-H1, and CDR-H3) and 20 natural amino acid residues(CDR-H3) into a combinatorial library are generally known, for example,by amplifying VH sequence by PCR, and/or performing random mutagenesisin CDR3.

The libraries disclosed herein can be used to screen for “ultra”humanized antibodies, in particular antibodies where human germlineresidues are incorporated into non-human donor CDRs. Accordingly, alsoprovided herein is humanized antibody or antigen-binding fragmentthereof that binds to a target antigen, wherein human germline residuesare incorporated into CDRs.

In certain embodiments, the human germline VL framework is the frameworkof DPK9 (IMGT name: IGKV1-39), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 13 SEQ ID NO. Light Chain 410 CDR-L1 RASQSISSYLN 411 CDR-L2AASSLQS 412 CDR-L3 QQSYSTP

In certain embodiments, the human germline VL framework is the frameworkof DPK12 (IMGT name: IGKV2D-29), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 14 SEQ ID NO. Light Chain 413 CDR-L1 KSSQSLLHSDGKTYLY 414 CDR-L2EVSNRFS 415 CDR-L3 MQSIQLP

In certain embodiments, the human germline VL framework is the frameworkof DPK18 (IMGT name: IGKV2-30), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 15 SEQ ID NO. Light Chain 416 CDR-L1 RSSQSLVYSDGNTYLN 417 CDR-L2KVSNRDS 418 CDR-L3 MQGTHWP

In certain embodiments, the human germline VL framework is the frameworkof DPK24 (IMGT name: IGKV4-1), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 16 SEQ ID NO. Light Chain 419 CDR-L1 KSSQSVLYSSNNKNYLA 420 CDR-L2WASTRES 421 CDR-L3 QQYYSTP

In certain embodiments, the human germline VL framework is the frameworkof HK102_V1 (IMGT name: IGKV1-5), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 17 SEQ ID NO. Light Chain 422 CDR-L1 RASQSISSWLA 423 CDR-L2DASSLES 424 CDR-L3 QQYNSYS

In certain embodiments, the human germline VL framework is the frameworkof DPK1 (IMGT name: IGKV1-33), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 18 SEQ ID NO. Light Chain 425 CDR-L1 QASQDISNYLN 426 CDR-L2DASNLET 427 CDR-L3 QQYDNLP

In certain embodiments, the human germline VL framework is the frameworkof DPK8 (IMGT name: IGKV1-9), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 19 SEQ ID NO. Light Chain 428 CDR-L1 RASQGISSYLA 429 CDR-L2AASTLQS 430 CDR-L3 QQLNSYP

In certain embodiments, the human germline VL framework is the frameworkof DPK21 (IMGT name: IGKV3-15), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 20 SEQ ID NO. Light Chain 431 CDR-L1 RASQSVSSNLA 432 CDR-L2GASTRAT 433 CDR-L3 QQYNNWP

In certain embodiments, the human germline VL framework is the frameworkof Vg_38K (IMGT name: IGKV3-11), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 21 SEQ ID NO. Light Chain 434 CDR-L1 RASQSVSSYLA 435 CDR-L2DASNRAT 436 CDR-L3 QQRSNWP

In certain embodiments, the human germline VL framework is the frameworkof DPK22 (IMGT name: IGKV3-20), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 22 SEQ ID NO. Light Chain 437 CDR-L1 RASQSVSSSYLA 438 CDR-L2GASSRAT 439 CDR-L3 QQYGSSP

In certain embodiments, the human germline VL framework is the frameworkof DPK15 (IMGT name: IGKV2-28), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 23 SEQ ID NO. Light Chain 440 CDR-L1 RSSQSLLHSNGYNYLD 441 CDR-L2LGSNRAS 442 CDR-L3 MQALQTP

In certain embodiments, the human germline VL framework is the frameworkof DPL16 (IMGT name: IGLV3-19), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 24 SEQ ID NO. Light Chain 443 CDR-L1 QGDSLRSYYAS 444 CDR-L2GKNNRPS 445 CDR-L3 NSRDSSGNH

In certain embodiments, the human germline VL framework is the frameworkof DPL8 (IMGT name: IGLV1-40), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 25 SEQ ID NO. Light Chain 446 CDR-L1 TGSSSNIGAGYDVH 447 CDR-L2GNSNRPS 448 CDR-L3 QSYDSSLSG

In certain embodiments, the human germline VL framework is the frameworkof V1-22 (IMGT name: IGLV6-57), and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody:

TABLE 26 SEQ ID NO. Light Chain 449 CDR-L1 TRSSGSIASNYVQ 450 CDR-L2EDNQRPS 451 CDR-L3 QSYDSSN

In certain embodiment, the human germline VL framework is the frameworkof human Vλ consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 27 SEQ ID NO. Light Chain 452 CDR-L1 TGSSSGGSYYVS or 453TGSSSDVGGSYYVS 454 CDR-L2 ENDSNRPS or 455 EDSNR(S/D)K(Q/G)QKPS 456CDR-L3 QSWDSSA(N/T) or 457 QSWDSSA(N/T)F(F/V)(G/V)

In certain embodiment, the human germline VL framework is the frameworkof human Vλ1 consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 28 SEQ ID NO. Light Chain 458 CDR-L1 SGSSSNIGNN(A/Y)V(N/H/S) or459 SGSSSNIIGNN(A/Y)V(N/H/S) 460 CDR-L2 GNN(K/N/Q)RPS 461 CDR-L3AAWDDSL(N/S)G

In certain embodiment, the human germline VL framework is the frameworkof human Vλ3 consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 29 SEQ ID NO. Light Chain 462 CDR-L1 CSGD(A/V)LG(K/S)KYAH 463CDR-L2 KDSERPS 464 CDR-L3 QSWDSSG(N/D/T/A) or 465 QSWDSSG(N/D/T/A)H

In certain embodiment, the human germline VL framework is the frameworkof human VK consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps.

TABLE 30 SEQ ID NO. Light Chain 466 CDR-L1 RASQSLLHSDGISSYLA or 467RASQGISSYLA 468 CDR-L2 AASSRAS 469 CDR-L3 QQYNSYP

In certain embodiment, the human germline VL framework is the frameworkof human VK1 consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. At positions where there is no consensus, residues in ( ) arethose that are tied for the most frequent residues.

TABLE 31 SEQ ID NO. Light Chain 470 CDR-L1 RASQGIS(N/S)YLA 471 CDR-L2AASSLQS 472 CDR-L3 QQYNSYP

In certain embodiment, the human germline VL framework is the frameworkof human VK2 consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 32 SEQ ID NO. Light Chain 473 CDR-L1 RSSQSLLHSDGNTYLD or 474RSSQSLLHSDDGNTYLD 475 CDR-L2 (K/T)(V/I)SNR(A/F)S 476 CDR-L3 MQATQFP

In certain embodiment, the human germline VL framework is the frameworkof human VK3 consensus sequence, and for each position within CDR-L1,CDR-L2, and CDR-L3, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. At positions where there is no consensus, residues in ( ) arethose that are tied for the most frequent residues.

TABLE 33 SEQ ID NO. Light Chain 477 CDR-L1 RASQS(S/V)(S/V)SSYLA 478CDR-L2 GASTRAT 479 CDR-L3 QQY(S/N/G/H)NWP

In certain embodiments, the human germline VH framework is the frameworkof DP54 or IGHV3-7, and for each position within CDR-H1, and CDR-H2, theresidue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 34 SEQ ID NO. Heavy Chain 480 CDR-H1 GFTFSSYWMS 481 CDR-H2ANIKQDGSEKYYVDSVKG

In certain embodiments, the human germline VH framework is the frameworkof DP47 or IGHV3-23 and for each position within CDR-H1, and CDR-H2, theresidue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 35 SEQ ID NO. Heavy Chain 482 CDR-H1 GFTFSSYAMS 483 CDR-H2AISGSGGSTYYADSVKG

In certain embodiments, the human germline VH framework is the frameworkof DP71 or IGHV4-59 and for each position within CDR-H1, and CDR-H2, theresidue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 36 SEQ ID NO. Heavy Chain 484 CDR-H1 GGSISSYYWS 485 CDR-H2GYIYYSGSTNYNPSLKS

In certain embodiments, the human germline VH framework is the frameworkof DP75 or IGHV1-2_02 and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 37 SEQ ID NO. Heavy Chain 486 CDR-H1 GYTFTGYYMH 487 CDR-H2GWINPNSGGTNYAQKFQG

In certain embodiments, the human germline VH framework is the frameworkof DP10 or IGHV1-69 and for each position within CDR-H1, and CDR-H2, theresidue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 38 SEQ ID NO. Heavy Chain 488 CDR-H1 GGTFSSYAIS 489 CDR-H2GGIIPIFGTANYAQKFQG

In certain embodiments, the human germline VH framework is the frameworkof DP7 or IGHV1-46, and for each position within CDR-H1, and CDR-H2, theresidue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 39 SEQ ID NO. Heavy Chain 490 CDR-H1 GYTGTSYYMH 491 CDR-H2GIINPSGGSTSYAQKFQG

In certain embodiments, the human germline VH framework is the frameworkof DP49 or IGHV3-30, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 40 SEQ ID NO. Heavy Chain 492 CDR-H1 GFTFSSYGMH 493 CDR-H2AVISYDGSNKYYADSVKG

In certain embodiments, the human germline VH framework is the frameworkof DP51 or IGHV3-48, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 41 SEQ ID NO. Heavy Chain 494 CDR-H1 GFTFSSYSMN 495 CDR-H2SYISSSSSTIYYADSVKG

In certain embodiments, the human germline VH framework is the frameworkof DP38 or IGHV3-15, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 42 SEQ ID NO. Heavy Chain 496 CDR-H1 GFTFSNAWMS 497 CDR-H2GRIKSKTDGGTTDYAAPVKG

In certain embodiments, the human germline VH framework is the frameworkof DP79 or IGHV4-39, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 43 SEQ ID NO. Heavy Chain 498 CDR-H1 GGSISSSSYYWG 499 CDR-H2GSIYYSGSTYYNPSLKS

In certain embodiments, the human germline VH framework is the frameworkof DP78 or IGHV4-30-4, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 44 SEQ ID NO. Heavy Chain 500 CDR-H1 GGSISSGDYYWS 501 CDR-H2GYIYYSGSTYYNPSLKS

In certain embodiments, the human germline VH framework is the frameworkof DP73 or IGHV5-51, and for each position within CDR-H1, and CDR-H2,the residue is either the respective human residue shown below, or itscorresponding residue from the non-human donor antibody:

TABLE 45 SEQ ID NO. Heavy Chain 502 CDR-H1 GYSFTSYWIG 503 CDR-H2GIIYPGDSDTRYSPSFQG

In certain embodiments, the human germline VH framework is the frameworkof human VH germline consensus sequence and for each position withinCDR-H1, and CDR-H2, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 46 SEQ ID NO. Heavy Chain 504 CDR-H1 GFTFSSYAM(H/S) or 505GFTFSSYAM(H/S)WS 506 CDR-H2 GWISPNGGSTYYADSVKG or 507GWISPKANGGSTYYADSVKG

In certain embodiments, the human germline VH framework is the frameworkof human VH3 germline consensus sequence, and for each position withinCDR-H1, and CDR-H2, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. Alternative sequences are provided for the consensus sequencewith and without gaps. At positions where there is no consensus,residues in ( ) are those that are tied for the most frequent residues.

TABLE 47 SEQ ID NO. Heavy Chain 508 CDR-H1 GFTFSSYAMS 509 CDR-H2SVISSDG(G/S)STYYADSVKG or 510 SVISSKADG(G/S)STYYADSVKG

In certain embodiments, the human germline VH framework is the frameworkof human VH5 germline consensus sequence, and for each position withinCDR-H1, and CDR-H2, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. At positions where there is no consensus, residues in ( ) arethose that are tied for the most frequent residues.

TABLE 48 SEQ ID NO. Heavy Chain 511 CDR-H1 GYSFTSYWI(S/G/H) 512 CDR-H2G(R/I/S)IYPGDSDTRYSPSFQG

In certain embodiments, the human germline VH framework is the frameworkof human VH1 germline consensus sequence, and for each position withinCDR-H1, and CDR-H2, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. At positions where there is no consensus, residues in ( ) arethose that are tied for the most.

TABLE 49 SEQ ID NO. Heavy Chain 513 CDR-H1 GYTFTSY(A/Y)(I/M)H 514 CDR-H2GWINP(G/Y)NGNTNYAQKFQ

In certain embodiments, the human germline VH framework is the frameworkof human VH4 germline consensus sequence, and for each position withinCDR-H1, and CDR-H2, the residue is either the respective human residueshown below, or its corresponding residue from the non-human donorantibody. At positions where there is no consensus, residues in ( ) arethose that are tied for the most frequent residues.

TABLE 50 SEQ ID NO. Heavy Chain 515 CDR-H1 GGSISSG(N/Y)YYWS 516 CDR-H2GYIYYSGSTYYNPSLKS

For example, if the framework sequence of human germline DPK9 is used asan acceptor for humanization, and the non-human donor CDR-L1 sequence isRASQDVGIYVN (SEQ ID NO: 2), then the CDR-L1 of the resulting humanizedantibody or antigen-binding fragment should be:RASQ(S/D)(IN)(S/G)(S/I)Y(L/V)N(SEQ ID NO:518). If the framework sequenceof human germline DPL16 is used as an acceptor for humanization, and thenon-human donor CDR-L1 sequence is RASQDVGIYVN (SEQ ID NO: 2), then theCDR-L1 of the resulting humanized antibody or antigen-binding fragmentshould be: (Q/R)(G/A)(D/S)(S/Q)(L/D)(RN)(S/G)(Y/I)Y(AN)(S/N) (SEQ IDNO:518). Under this design rationale, once a specific human germlinesequence is selected as an acceptor, then five of the six CDR can bereadily designed, as each individual position generally only has twochoices—the germline residue from the same human germline, or thecorresponding donor residue.

As shown in the Examples, certain positions in CDRs prefer non-humandonor residues, whereas certain positions in CDRs tolerate humangermline residues well. Positions that generally tolerate human germlineresidues well are candidates for CDR humanization. For example, as shownin FIG. 10, the n-terminal 4 residues of the VK CDR1, and last 6residues of the VH CDR2 showed low retention rate of non-human donorresidues. This indicates that these residues are candidates for CDRhumanization.

Accordingly, provided herein are humanized antibodies or antigen-bindingfragment thereof (such as an antibody variable domain), and librariescomprising such antibodies or antigen-binding fragment thereof (such asan antibody variable domain), wherein one or more of the n-terminal 4residues of CDR-L1 (residues 24, 25, 26, and 27 respectively, based onKabat numbering) comprise the corresponding human germline residues. Incertain embodiments, the light chain framework sequence is from a humanVK germline.

Also provided herein are humanized antibodies or antigen-bindingfragment thereof (such as an antibody variable domain), and librariescomprising such antibodies or antigen-binding fragment thereof (such asan antibody variable domain), wherein one or more of the last 6 residuesof CDR-H2 (residues 60, 61, 62, 63, 64, 65 respectively, based on Kabatnumbering) comprise the corresponding human germline residues. Incertain embodiments, the heavy chain framework sequence is from a humanVH3 germline, VH1 germline, VH5 germline, or VH4 germline.

D. Antibody Display

The antibody libraries described herein are generally screened toidentify specific clones that show desired antigen-binding affinities,or other properties (e.g., potency). For screening, a variety oftechniques may be used to display these antibody variable domains.

Commonly used display libraries include, e.g., phage display, yeastdisplay, mammalian cell surface display, bacterial display, viraldisplay, mRNA display, ribosome display or DNA display library.

One exemplary display library is a phage display library. Phage displayis a technique by which variant polypeptides are displayed as fusionproteins to a coat protein on the surface of phage, e.g., filamentousphage, particles. One advantage of phage display library is that largelibraries of randomized protein variants can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Polyvalent phage display methods have been used for displayingsmall random peptides and small proteins through fusions to either geneIII or gene VIII of filamentous phage. Wells and Lowman, Curr OpinStruct Biol, 3:355-362 (1992). In monovalent phage display, a protein orpeptide library is fused to a gene III or a portion thereof, andexpressed at low levels in the presence of wild type gene III protein sothat phage particles display one copy or none of the fusion proteins.Avidity effects are reduced relative to polyvalent phage so that sortingis on the basis of intrinsic ligand affinity, and phagemid vectors areused, which simplify DNA manipulations. Lowman and Wells, Methods: Acompanion to Methods in Enzymology, 3:205-0216 (1991).

A phagemid is a plasmid vector having a bacterial origin of replication,e.g., ColE1, and a copy of an intergenic region of a bacteriophage. Thephagemid may be used on any known bacteriophage, including filamentousbacteriophage and lambdoid bacteriophage. The plasmid will alsogenerally contain a selectable marker for antibiotic resistance.Segments of DNA cloned into these vectors can be propagated as plasmids.When cells harboring these vectors are provided with all genes necessaryfor the production of phage particles, the mode of replication of theplasmid changes to rolling circle replication to generate copies of onestrand of the plasmid DNA and package phage particles. The phagemid mayform infectious or non-infectious phage particles. Phagemids may containa phage coat protein gene or fragment thereof linked to a heterologouspolypeptide gene as a gene fusion such that the heterologous polypeptideis displayed on the surface of the phage particle.

A phage vector is a double stranded replicative form of a bacteriophagecontaining a heterologous gene and capable of replication. The phagevector has a phage origin of replication allowing phage replication andphage particle formation. The phage is preferably a filamentousbacteriophage, such as an M13, f1, fd, Pf3 phage or a derivativethereof, or a lambdoid phage, such as lambda, phage 21, phi80, phi81,82, 424, 434, or a derivative thereof.

A phage vector may also encode a tag, for example, a polyhistidine tag,to facilitate the detection or identification of antibody variabledomains that bind to a specific antigen.

4. Screening and Selection of Antibodies

The displayed antibody variable domains can then be screened for, e.g.,the ability to bind the target antigen. For example, the target antigencan be attached with a detectable moiety, such as biotin. Polypeptidesthat bind to the target antigen can be separated from unbound ones by amolecule that binds to the detectable moiety, such asstreptavidin-coated beads where biotin is the detectable moiety.Affinity of binders (polypeptide that binds to target) can be determinedbased on concentration of the target molecule used, using formulas andbased on criteria known in the art.

The target antigen may also be attached to a suitable matrix such asagarose beads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyalkyl methacrylate gels, polyacrylic andpolymethacrylic copolymers, nylon, neutral and ionic carriers, and thelike. After attachment of the target antigen to the matrix, theimmobilized target is contacted with the antibody library. Polypeptidesbound to the immobilized antigen can then be separated from those thatdo not bind to the target by washing.

The binders can be isolated and then re-amplified or expressed in a hostcell, and subjected to additional rounds of selection for binding oftarget molecules. Any number of rounds of selection or sorting can beutilized.

In certain embodiments, the library is screened to select a polypeptidethat binds to the target antigen, with an affinity (Kd) value of no morethan about 1×10⁻³ M, such as no more than about 1×10⁻³ M, no more thanabout 9×10⁻⁴ M, no more than about 8×10⁻⁴ M, no more than about 7×10⁻⁴M, no more than about 6×10⁻⁴ M, no more than about 5×10⁻⁴M, no more thanabout 4×10⁻⁴ M, no more than about 3×10⁻⁴ M, no more than about 2×10⁻⁴M, no more than about 1×10⁻⁴ M, no more than about 9×10⁻⁵ M, no morethan about 8×10⁻⁵ M, no more than about 7×10⁻⁵ M, no more than about6×10⁻⁵ M, no more than about 5×10⁻⁵ M, no more than about 4×10⁻⁵ M, nomore than about 3×10⁻⁵ M, no more than about 2×10⁻⁵ M, no more thanabout 1×10⁻⁵ M, no more than about 9×10⁻⁶ M, no more than about 8×10⁻⁶M, no more than about 7×10⁻⁶ M, no more than about 6×10⁻⁶ M, no morethan about 5×10⁻⁶ M, no more than about 4×10⁻⁶ M, no more than about3×10⁻⁶ M, no more than about 2×10⁻⁶ M, no more than about 1×10⁻⁶ M, nomore than about 9×10⁻⁷ M, no more than about 8×10⁻⁷ M, no more thanabout 7×10⁻⁷ M, no more than about 6×10⁻⁷ M, no more than about 5×10⁻⁷M, no more than about 4×10⁻⁷ M, no more than about 3×10⁻⁷ M, no morethan about 2×10⁻⁷ M, no more than about 1×10⁻⁷ M, no more than about9×10⁻⁸ M, no more than about 8×10⁻⁸ M, no more than about 7×10⁻⁸M, nomore than about 6×10⁻⁸ M, no more than about 5×10⁻⁸ M, no more thanabout 4×10⁻⁸ M, no more than about 3×10⁻⁸ M, no more than about 2×10⁻⁸M, no more than about 1×10⁻⁸ M, no more than about 9×10⁻⁹ M, no morethan about 8×10⁻⁹ M, no more than about 7×10⁻⁹ M, no more than about6×10⁻⁹ M, no more than about 5×10⁻⁹ M, no more than about 4×10⁻⁹ M, nomore than about 3×10⁻⁹ M, no more than about 2×10⁻⁹ M, no more thanabout 1×10⁻⁹ M, from about 1×10⁻³ M to about 1×10⁻¹³ M, 1×10⁻⁴ M toabout 1×10⁻¹³ M, 1×10⁻⁵M to about 1×10⁻¹³ M, from about 1×10⁻⁶ M toabout 1×10⁻¹³M, from about 1×10⁻⁷ M to about 1×10⁻¹³ M, from about1×10⁻⁸ M to about 1×10⁻¹³ M, from about 1×10⁻⁹ M to about 1×10⁻¹³ M,1×10⁻³ M to about 1×10⁻¹² M, 1×10⁻⁴ M to about 1×10⁻¹² M, from about1×10⁻⁵ M to about 1×10⁻¹² M, from about 1×10⁻⁶ M to about 1×10⁻¹² M,from about 1×10⁻⁷ M to about 1×10⁻¹² M, from about 1×10⁻⁸ M to about1×10⁻¹² M, from about 1×10⁻⁹ M to about 1×10⁻¹² M, 1×10⁻³ M to about1×10¹¹ M, 1×10⁻⁴M to about 1×10¹¹ M, from about 1×10⁻⁵ M to about 1×10¹¹M, from about 1×10⁻⁶ M to about 1×10⁻¹¹ M, from about 1×10⁻⁷ M to about1×10⁻¹¹ M, from about 1×10⁻⁸ M to about 1×10¹¹ M, from about 1×10⁻⁹ M toabout 1×10⁻¹¹ M, 1×10⁻³ M to about 1×10⁻¹⁰ M, 1×10⁻⁴M to about 1×10⁻¹⁰M, from about 1×10⁻⁵ M to about 1×10⁻¹⁰ M, from about 1×10⁻⁶ M to about1×10⁻¹⁰ M, from about 1×10⁻⁷ M to about 1×10⁻¹⁰ M, from about 1×10⁻⁸M toabout 1×10¹⁰ M, or from about 1×10⁻⁹M to about 1×10¹⁰ M.

In certain embodiments, a polypeptide that binds to the target antigenwith a binding affinity (Kd) value that is equal or less than thebinding affinity (Kd) value of the original non-human donor antibody.

Although in general, Kd at nanomolar range is desired, in certainembodiments, low affinity antibodies may be preferred, for example, fortargeting highly expressed receptors in compartments and avoidingoff-target binding. Further, some therapeutic applications may benefitfrom an antibody with lower binding affinity to facilitate antibodyrecycling.

In certain embodiments, the selected antibody variable domains may alsobe further screened by other biological activity assays, e.g., in orderto evaluate its potency, pharmacological activity, and potentialefficacy as a therapeutic agent. Such assays are known in the art anddepend on the target antigen and intended use for the antibody. Examplesinclude e.g., tumor cell growth inhibition assays; antibody-dependentcellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC)assays; agonistic activity or antagonist activity assays.

Once a desired clone is selected, the sequence of the antibody variabledomain, and nucleic acid encoding such antibody variable domain, can bedetermined using standard sequencing techniques. Nucleic acid sequenceencoding a desired antibody variable domain may be inserted into othervectors (such as cloning and expression vectors) for recombinantproduction and characterization.

Suitable cloning and expression vectors can include a variety ofcomponents, such as promoter, enhancer, and other transcriptionalregulatory sequences. The vector may also be constructed to allow formovement of antibody variable domain between different vectors.

Selected antibody (or antigen-binding fragment thereof) may be maderecombinantly produced using a suitable host cell. Nucleic acid encodingthe antibody or antigen-binding fragment thereof can be cloned into anexpression vector, which can then be into a host cell, such as E. colicell, a yeast cell, an insect cell, a simian COS cell, a Chinese hamsterovary (CHO) cell, or a myeloma cell that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells.

Antibody fragments can be produced by proteolytic or other degradationof the antibodies, by recombinant methods, or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable.

The selected antibody or antigen-binding fragment thereof may beaffinity-matured. For example, affinity matured antibodies can beproduced by procedures known in the art (Marks et al., 1992,Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci,USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al.,1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol.,154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; andWO2004/058184).

5. Formulations and Uses

Antibodies or antigen-binding fragments identified from the librarydescribed herein can be formulated as pharmaceutical formulations. Thepharmaceutical formulation may further comprise pharmaceuticallyacceptable carriers, excipients, or stabilizers (Remington: The Scienceand practice of Pharmacy 20th Ed., 2000, Lippincott Williams andWilkins, Ed. K. E. Hoover), in the form of lyophilized formulations oraqueous solutions. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations, and maycomprise buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

The antibodies or antigen-binding fragments identified from the librarydescribed herein can be used for therapeutic, diagnostic, ornon-therapeutic purposes. For example, the antibody or antigen-bindingfragment thereof may be used as an affinity purification agents (e.g.,for in vitro purification), as a diagnostic agent (e.g., for detectingexpression of an antigen of interest in specific cells, tissues, orserum)

For therapeutic applications, antibodies or antigen-binding fragmentsidentified from the library described herein can be administered to amammal, especially a human by conventional techniques, such asintravenously (as a bolus or by continuous infusion over a period oftime), intramuscularly, intraperitoneally, intra-cerebrospinally,subcutaneously, intra-articularly, intrasynovially, intrathecally,orally, topically, or by inhalation. The antibodies or antigen-bindingfragments also are suitably administered by intra-tumoral, peri-tumoral,intra-lesional, or peri-lesional routes. The antibodies orantigen-binding fragments can be used in prophylactic treatment ortherapeutic treatment.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Current evidence suggests that the main risk factors for antibodyimmunogenicity in man are human t-cell epitope content and, to a lesserextent, t-cell independent b-cell responses. B-cell epitopes arechallenging to predict and b-cell-only responses to biotherapeuticsappear to be driven by protein aggregates. Important factors in reducingantibody immunogenicity risk in the clinic are low t-cell epitopecontent, minimized non-human germline content and low aggregationpotential.

Examples provided herein describe the “Augmented Binary Substitution”design principle that generates stable, soluble, ultra-humanizedantibodies via single-step CDR redundancy minimization. For threeantibodies from three separate key immune host species, binarysubstitution CDR cassettes were inserted into preferred human frameworksto form libraries in which only the donor (non-human) residue or humangermline destination residue was encoded at each position. The CDR-H3 ineach case was also augmented with 1±1 random substitution per clone.Each library was then screened for clones with restored antigen bindingcapacity. Lead ultra-humanized clones demonstrated high stability, withaffinity and specificity equivalent to, or better than, the parentalimmunoglobulin. Significantly, this was mainly achieved on germlineframeworks by simultaneously subtracting up to 19 redundant non-humanresidues in the CDRs. This significantly lowered non-human sequencecontent, minimized t and b-cell epitope risk in the final molecules andprovided a heat map for desired non-human CDR residue content of eachantibody.

Example 1 Library Design, Build and Characterization

Rat anti-RAGE XT-M4, rabbit anti-A33, and chicken anti-pTaupT231/pS235_1 IgGs were generated on the human IgG1 backbone with eitherparental (Par-RAGE, Par-A33 or Par-pTau), grafted (Graft-RAGE, Graft-A33or Graft-pTau), or classically humanized (CL-Hum-RAGE, CL-Hum-A33)v-domains. In scFv format, the parental form of each of these antibodiesretained antigen binding, while the human FW-grafted versionsdemonstrated little to no binding (FIGS. 5A-5C). ABS ultra-humanizationlibraries (ABS-RAGE, ABS-A33, ABS-pTau) were constructed (FIG. 1) togenerate 1.8×10⁹ independent clones for ABS-RAGE, 1.1×10¹⁰ for ABS-A33and 4.9×10⁹ for ABS-pTau (theoretical binary diversity for ABS-RAGE is2²⁷ positions=1.34×10⁸, for ABS-A33 232=4.29×109 and for ABS-pTau2³³=8.59×10⁹).

The quality of pTau library build was verified by sequence analyses of≧96 clones/library. After library transformation, the full scFv insertsequences were obtained for 96 clones, via sanger sequencing. Positionsmutated in the CDRs show the expected (approximately 50:50) variabilityat all positions expected to be sampled by binary substitutions andlow-level mutagenesis in the CDR-H3, confirming the integrity of thesampled library. <1% of clones contained out of frame or truncatedinserts. Libraries were rescued using helper phage M13 and selectionsperformed on their cognate targets.

Example 2 Identification and Analysis of Ultra-Humanized Clones

Clone selection in ABS library screening (pTau example) was conducted.Periprep ELISA was conducted to screen for single clones picked frommultiple rounds of phage display selections of the ABS-pTau library. Onehundred and eighty-eight clones were prioritized on the basis ofretention of binding to the pT231_pS253 phosphopeptide, with A450readings above the negative control (Anti-RAGE scFv), and equivalent toor above that of Par-pTau scFv. Periprep HTRF was conducted to screenthese 188 single clones for epitope competition with wild-type IgG.Clones were prioritized on the basis of neutralisation of Par-pTau IgGbinding to the pT231_pS253 phosphopeptide, with % ΔF readings lower thanthe negative control (Anti-RAGE scFv) and equivalent to or better thatof Par-pTau scFv.

Post-selection screening revealed the presence of numerous scFv cloneswith significantly increased human content within the CDRs. In theABS-RAGE and ABS-A33 leads, the FW sequences remained fully germline. Inthe ABS-pTau leads, all selected clones retained the T46 back-mutation,illustrating that this VL-FW2 residue is desired to humanize chickenantibodies (FIGS. 6A-6B). Human germline amino acid content wasquantified within the CDRs of parental antibodies and ABS leads andexpressed as a percentage (Table 1). Human content had raised 17-29% ineach case. From top to bottom, CDRL1: SEQ ID NOs. 278-290; CDRL2: SEQ IDNOs. 291-303; CDRL3: SEQ ID NOs. 304-316; CDRH1: SEQ ID NOs. 317-329;CDRH2: SEQ ID NOs. 330-342; CDRH3: SEQ ID NOs. 343-352.

Example 3 Lead IgG Affinity, Stability and Specificity Characteristics

ABS leads in human IgG1 format were analyzed for specificity andstability. HTRF data (FIGS. 7A-7C), showed that the lead ABS-derivedIgGs had successfully maintained full epitope competition with theirrespective parental clones (Table 1). Biacore analyses showedapproximately 2-fold affinity improvements for C7-ABS-RAGE andC21-ABS-pTau over Par-RAGE and Par-pTau, respectively, while C2-ABS-A33maintained equivalent affinity to Par-A33 (Table 1).

A baculovirus ELISA assay (FIG. 8A) has been reported in antibodypolyreactivity screening as a risk indicator for poor pK in vivo. Inthis assay, no reactivity was observed for any of the RAGE, A33 or pTauclones in comparison to an internal positive control antibody. For theanti-RAGE and anti-A33 antibodies, a high-sensitivity Biacore assay wasalso established, to examine the possibility that v-gene engineeringmight lead to low affinity interactions with multiple classes ofproteins. A panel of 18 fully-purified, recombinant, non-target proteinswas examined. This method showed that C7-ABS-RAGE and C2-ABS-A33 bothmaintained highly specific binding to their respective antigens (FIGS.8B and 8C). For the anti-pTau antibodies, specificity for pT231/pS235was confirmed using Biacore assays (FIG. 8D). All ABS-derived leads inthis study were, therefore, absent of the ‘charge asymmetry’,lipophilicity, off-target protein binding or other problems that canarise during v-gene engineering.

DSC analysis of IgG thermal stabilities demonstrated that C7-ABS-RAGE,C2-ABS-A33 and C21-ABS-pTau were highly stable. C7-ABS-RAGE wasparticularly thermostable with a Fab Tm of 85° C.; similar toGraft-RAGE, but almost 8° C. higher than that of the CL-Hum-RAGE (FIG.2A, Table 1). This is a finding of note, as it highlighted that thepresence of back-mutations in CL-Hum-RAGE had significantly decreasedthe stability of the v-domains in comparison to the highly stable graft.C21-ABS-pTau exhibited a Fab Tm of 70° C., 4° C. higher than Graft-pTau(FIG. 2B). In forced aggregation analyses C7-ABS-RAGE, C21-ABS-pTau andC2-ABS-A33 all showed <1% aggregation at 60° C. (FIG. 2D, E, F, Table1). Graft-pTau, in contrast, exhibited >90% aggregation at 60° C.Importantly, analysis of pH shock tolerance (which mimics virus-killingpH hold in mAb manufacturing) also showed each of the IgGs to be highlystable, with <3% loss observed.

Example 4 Human t and b-Cell Epitope Minimization in ABS Leads

ABS leads and associated precursors were examined for potential t-cellepitope content using the EpiMatrix software, generating a t-regitopeadjusted score for each clone (FIG. 3A), as suggested in the recentdraft FDA immunogenicity assessment guidelines. C7-ABS-RAGE, C2-ABS-A33and C21-ABS-pTau showed scores of −53.72, −55.22 and −67.33 points,respectively. This lowered the projected immunogenicity of all clonesinto the same range as antibodies such as trastuzumab, which has beenwell tolerated in the clinic, and lower than ‘fully human’ antibodiessuch as adalimumab (FIG. 3A).

Analysis at the individual peptide level predicted that t-cell epitopecontent was clearly reduced for C7-ABS-RAGE and C21-ABS-pTau incomparison to their respective parental forms (FIG. 9). T-regitopecontent was also increased. Notably, the ratio of t-cell epitopes tot-regitopes was improved in C7-ABS-RAGE in comparison to CL-Hum-RAGE.Indeed, the removal of the back mutation found in the VL FW2 ofCL-Hum-RAGE not only aided stabilization of the v-domains (FIG. 2A), butalso removes a t-cell epitope and converts it into a germline t-regitope(FIG. 9). Analysis of the sequence of C21-ABS-pTau showed that theABS-derived germlining at key positions in the CDR-H2 had ablated aforeign t-cell epitope at the N-terminus of the loop that had beenintroduced by CDR-grafting. Some potential epitopes of foreign sequencewere still present however, even after ultra-humanization (FIG. 9). Thisreflects the need to retain certain key contact residues in the paratopeand balance target recognition with t-cell epitopes and overall“humanness”. The L46T back mutation in C21-ABS-pTau was not predicted tointroduce a t-cell epitope and this clone retained only a singlepredicted foreign t-cell epitope (in comparison to 5 in Par-pTau),driven by Q33 in the V_(H).

As a surrogate for b-cell epitope availability, non-humansolvent-accessible surface area (nhSASA, measured in Å²) was calculatedfor the parental, graft and ABS lead clones. Clones C7-ABS-RAGE,C2-ABS-A33 and C21-ABS-pTau demonstrated minimized non-human surfacearea (FIG. 3B). C7-ABS-RAGE exhibited a reduction in nhSASA to 1077.6Å², in comparison to the Par-RAGE and Graft-RAGE at 3084.8 and 1957.6,respectively. This represents a 45% reduction even in comparison to theGraft-RAGE. C21-ABS-pTau demonstrated a reduction to 1009.0 Å², incomparison to the Par-pTau and Graft-pTau at 3365.3 and 1372.1,respectively. C2-ABS-A33 showed a reduction to 1583.9 Å², in comparisonto the Par-pTau and Graft-pTau at 4260.7 and 2240.2, respectively (FIG.3B).

Further analyses were performed using publically available software, tonumerically define the overall levels of human repertoire identity ofthe parental and ABS-derived leads, in comparison with 33 antibodiescurrently approved as therapeutics with murine, humanized or “fullyhuman” v-domains. These analyses showed that the ABS clones haddistinctly improved T20, G and Z scores over parental clones. Indeed,the C7-ABS-RAGE clone had scores placing it in the range of values foundfor the ‘fully human’ antibody group, with the C2-ABS-A33 andC21-ABS-pTau clones close behind (FIG. 3C).

Example 5 Strongly Maintained Non-Human CDR Content Via MutationalTolerance and Structural Analyses

The screening of output clones from the ABS-pTau library identified 188sequence-unique hits with binding signals ≧the parental scFv (data notshown). For residues targeted in the library for binary substitution,positional amino acid usage frequencies were calculated for these hitsand expressed as a percentage (FIGS. 4A-4B). Amino acid positions withparental residue usage frequencies of >75% were labeled “stronglymaintained” (SM), i.e. with the chicken residue being positivelyselected. Those with frequencies below 25% were labeled “stronglydeleted” (SD), i.e. the human germline residue being preferred.

Heatmaps of non-human residue content in the anti-pTau binding site forPar-pTau, Graft-pTau and C8-ABS-pTau were also generated (data notshown). The heatmaps show that the ABS process better defines residuesthat may be important for antigen-binding function.

When the SM residues were compared with those previously predicted to bekey contacts via a co-crystal structure, the two populations were foundto clearly overlap. Across both chains, however, SM positions were foundto be only 29.6% (17/55) of the total CDR residues outside the CDR-H3.In the V_(H) domain, Q33, T52, S53, R54 were all predicted contactresidues and all were SM, with retention frequencies >90%. G55 and G56were also predicted to be key contacts but were not sampled in thelibrary, as they were fully conserved human to chicken. Interestingly,the S53G substitution, while not heavily favored in the selectedpopulation, could clearly be functional, as seen in the C21-ABS-pTauclone, so long as T52, R54, G55 and G56 were maintained (FIG. 6). OtherSM residues in the V_(H) were found to be contact-proximal and/ordesirable for appropriate presentation of the contact residues, such asM35, A49, G50, V57 and G59.

Of 4 predicted contact residues in the V_(L), only Y91 was found to beSM and was retained at 100%. Other SM residues were predominantly foundin stem-loop positions of CDR-L3 (G89, G96, G98) and CDR-L1 (G34), whichmay be influential on loop structure. Additionally, the SM N51 siteforms structurally supportive hydrogen bonds between the CDR-L1 andV_(L) FW2.

Out of 33 residues sampled by binary substitution, only a single SDresidue (V_(H)L29) was identified, suggesting that all 16 other non-SMresidues were interchangeable. On the basis of these analyses, weinterpreted the “strongly non-human CDR content”, meaning those parentalresidues that are unlikely be germlined, even if compensated by themutation of a residue elsewhere in the paratope (FIGS. 4A-4B). This alsoinformed the minimization of chicken residue content by making acombination clone, Combo-ABS-pTau. This clone contains the most “human”variant of each CDR that had been observed in the top ABS-pTau hits(FIG. 6, Table 1). This reduced non-human content by another 6 residuesversus C21-ABS-pTau, pushing the Combo-ABS-pTau to 76% human in theCDRs, but adding one more predicted t-cell epitope than C21-ABS-pTau(FIG. 9). The retained non-human residues in Combo-ABS-pTau closelymatched to the SM set of residues described above (FIGS. 4A-4B).Combo-ABS-pTau was found to be soluble, stable and maintained thebinding affinity and specificity of Par-pTau (Table 1, FIG. 2A).

Example 6 Additional CDR-Humanized Antibodies

The sequences of additional CDR-humanized antibodies are shown in Tables7-12. CDR sequences are in bold. Donor v-genes (murine, rat, rabbit, orchicken) and human germline DP/J designations are included. CDR residuesfrom the parent clone that differ from human germline are underlined.

SUMMARY

Despite considerable investigation, current antibody humanizationmethods often create therapeutic molecules with significant risk factorsfor the failure of a lead drug due to potential immunogenicity and/orpoor pK in the clinic, or because the molecule cannot be manufacturedand delivered in a cost-effective manner. These risks are potentiallyexacerbated if the lead is derived from hosts such as rats, rabbits orchickens, rather than the heavily characterized antibody repertoires ofmice and humans.

Antibodies from alternative immune species can provide excellent IgGswith unique functional characteristics against problematic targets (e.g.highly conserved across species), but their antibodies are also known toexhibit unique sequence/structural features. These antibodies thereforerequire maximal humanization and development validation if they are togain broad acceptance as potential clinical leads. Indeed, despite theirtherapeutic potential, there are currently no chicken antibodies andonly one known humanized rabbit antibody in the clinic. In establishingthe ABS technology we have shown that it is possible to minimizeclinical and manufacturing concerns, by making antibodies from all 3sources stable, soluble and of low immunogenicity risk. When analyzed insilico, human identity and t-cell epitope risk appeared to beindivisible between C7-ABS-RAGE and currently marketed ‘fully human’antibodies, with C21-ABS-pTau and C2-ABS-A33 comparable to the best ofthe humanized mouse antibodies currently approved for clinical use.

Other humanization methods do not factor in the CDRs themselves asmediators of stability and solubility, in addition to the frameworks.Antibodies from species with limited starting framework diversity inboth the V_(H) and V_(L) genes fit the ABS technology particularly well.Indeed, chickens and rabbits use V_(H) repertoires that are highlyhomologous to human V_(H)3 domain. For murine antibodies, FW diversityin the functional repertoire is much higher than for chickens orrabbits. Prior estimations of v-domain homology, pairing angle andV_(H)-V_(L) packing are therefore prudent, to aid the prediction ofwhether preferred germlines.

Previous methods that maintain the animal CDR-H3 (+/−CDR-L3), thensample human repertoire diversity to return binding affinity, may sufferfrom an inability to recapitulate the critical structuralcharacteristics found outside the CDR-H3s of non-murine antibodies, asexemplified by our anti-pTau mAb. These methods also frequently leave,or generate, significant numbers of framework mutations away fromgermline which can lower the stability of v-domains. Indeed, theC7-ABS-RAGE clone illustrated that the CDRs from XT-M4 could be heavilygermlined and the back mutations from classical humanization fullyremoved, greatly improving stability in the final molecule.

This study illustrates that 3 separate antibodies from 3 species,targeting 3 different epitopes, all have high levels of sequenceredundancy in their paratopes that can be exploited for v-domain riskreduction engineering without the need for prior structural analyses.The retention of SM residues in the CDRs of selected clones after ABSstrongly correlated with the prediction of key contact residues in theco-crystal structure of anti-pTau with its target antigen. Residues werealso found to be SM if they were likely to be desirable for the correctpresentation of CDR loops. In only one case was a framework backmutation necessary to include during humanization (V_(L) L46T,anti-pTau). This suggests that many of the back mutations requiredduring classical humanization of anti-RAGE and anti-A33 were likelynecessitated by the retention of non-human CDR residues that clash withhuman framework residue side chains, but are functionally redundant inantigen binding. ABS intrinsically minimized redundant animal-derivedCDR content by selecting for the retention of essential non-humanresidues and allowing the rest of the CDR to be converted to thesequence of the destination v-gene. This approach thereby simultaneouslyoptimized all functional parameters of these three potential therapeuticantibodies, which were derived from species often used in monoclonalantibody generation against challenging therapeutic targets.

Materials and Methods for Examples 1-6

ScFv-based library designs. Parental and CDR-grafted forms of ratAnti-RAGE, rabbit anti-A33 and chicken anti-pTau antibodies, plus aclassically humanized (CL-Hum) version of XT-M4 were synthesized(Geneart™) in V_(L)-V_(H) scFv format, ligated into the phagemid pWRIL-1and cloned into E. coli TG1 cells. Soluble periplasmic E. coliexpression was confirmed by SDS-PAGE and western blot. Function of eachconstruct was assessed via direct binding ELISA (as purified scFv orperiprep). Based on these scFv constructs, Augmented Binary Substitutionlibraries were designed in silico (FIG. 1) and synthesized as finisheddsDNA scFv fragments (Geneart™). Anti-pTau is a Type 1 chicken IgG withcritical secondary structural characteristics in CDR H2 and H3, and arecent structural study of a humanized chicken antibody suggested that aback mutation at V_(λ) FW2 position 46 (L46T) is critical to the correctpacking of the V_(λ) against the CDR-H3 stem-loopTo examine whether ornot this was still true when random point mutations are also beingsimultaneously sampled in the CDR-H3, a binary substitution (L/T) wasallowed at VA position 46 in the ABS-pTau library.

Construction, Selection and Screening of scFv Libraries.

The ABS scFv libraries were constructed rescued and selected. Solutionphase selection on biotinylated target antigen with streptavidin beadswas employed throughout. Post-selection ELISA and HTRF screening,epitope competition analyses and reformatting were performed. Fordetails, see Finlay, W. J. et al. J Mol Biol 388, 541-558 (2009).

IgG Expression and Biophysical Analyses.

IgGs were transiently expressed in HEK-293f cells after transfectionwith IgG expression plasmids and lipofectamine 2000 (Invitrogen),according to manufacturer's protocols. Automated purification wascarried out using ProPlus resin tips on the MEA system (Phynexus).Differential Scanning Calorimetry, Forced Aggregation and pH stabilityanalyses were performed according to King, A. C. et al. Protein Sci 20,1546-1557 (2011).

Biacore Analysis of Binding Kinetics.

Biacore analysis was performed using the T-200 biosensor, series S CM5chips, an amine-coupling kit, 10 mM sodium acetate immobilization bufferat pH 5.0, 10×HBS-P running buffer and NaOH for regeneration (GEHealthcare). Kinetic assay conditions were established to minimize theinfluence of mass transfer, avidity and rebinding events. A predefinedligand immobilization program was set up to immobilize approximately 100Response Units (RU) of IgG on the chip. Purified target proteins werediluted in HBS-P running buffer to a range of final concentrations andinjected at 50 μl/min for 3 mins. Dissociation was allowed to proceedfor 10 min followed by a 5 sec pulse of 20 mM NaOH for regeneration ofthe chip surface. All sensorgrams were analyzed using the Biacore T-200evaluation software.

Binding Specificity Analyses.

Anti-RAGE, pTau and A33 antibodies were tested for polyreactivity byELISA and Biacore analyses. ELISAs were performed against singlestranded DNA, double stranded DNA, insulin and lipopolysaccaride, andagainst Baculovirus particles All polyreactivity analyses used parentalantibodies and Pfizer in-house positive and negative control antibodies.

Biacore specificity analyses were performed using the T-200 biosensor,series S CM5 chips, an amine-coupling kit, 10 mM sodium acetateimmobilization buffer at pH 5.0, 10× HBS-P running buffer and NaOH forregeneration (GE Healthcare). A predefined ligand (IgG) immobilizationprogram was set up to immobilize approximately 300 Response Units (RU)on the flow cell for each IgG to be tested. For Anti-RAGE and anti-A33,a panel of fully-purified recombinant target and non-target antigenswere diluted in HBS-P running buffer to a final concentration of 500 nM.Four groups of antigens were examined, including: cell membrane proteins(mTRKB, hTRKB, mEGFR, hEGFR, hFceR1, hIL-21R, mICAM1, mICAM2, hICAM1,hCD33, hLAMP-1, hLOX-1, and), soluble signaling molecules (mTNFa, hVEGF,hCXCL13) and albumins (BSA, HSA and MSA). These proteins were injectedat 50 μl/min for 3 min, followed by a 5 sec pulse of 20 mM NaOH forregeneration of the chip surface. For pTau, a series of pTau-derivedphosphorylated and non-phosphorylated peptides were flowed, as in Shihet al.³. All sensorgrams were analyzed using the Biacore T-200evaluation software.

Modeling Analyses.

Variable domain structural models were generated for the parental,humanized and ABS humanized variants of the anti-pTAU and the anti-RAGEantibodies. The Protein Databank (PDB) crystal structure 4GLR of theanti-pTau antibody was used for the parental pTau model. For thehumanized and ABS humanized pTau antibodies, we generated homologymodels using Modeller version 9.12 with the PDB structures of 4GLR and3G6A as templates. For all three XTM4 structures, homology models werealso generated using Modeller with template structure 1fvd, 1dql, 3hns,1mhp, 1bbj, 1bog, 1aif, 1arl and 1rmf for the parental; 1fvd, 1dql,1mhp, 3hns, 1bbj, 1bog, 1aif, 1arl and 1 rmf for the humanized; and1mhp, 3hns, 2cmr, 1gig, 2ghw, 1aif and 1rmf for the ABS humanized. Thenon-human solvent accessible surface area (nhSASA) was calculated usingthe “Solvent Accessibility” calculator in the molecular modelingsoftware suite Discovery Studio Client 4.0 (Accelrys Inc). The nhSASAwas defined as the sum of the side-chain SASA of residues that were notidentical to germline.

In Silico t-Cel Epitope Assessment.

Sequences of antibody V_(H) and V_(L) regions were analyzed by EpiMatrix(Epivax, RI) Briefly, each domain was parsed into overlapping 9-merpeptides with each peptide overlapping the last by eight amino acids.Each peptide was then scored for predicted binding to each of eight HLAClass II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801,DRB1*1101, DRB1*1301, and DRB1*1501) which represent HLA supertypescovering 97% of human populations worldwide. Any peptide scoring above1.64 on the EpiMatrix “Z” scale (approximately the top 5% of the randompeptide set) was classed as a “hit” for binding to the MHC molecule forwhich it was predicted. Peptides scoring four or more hits from theeight alleles predicted are considered as possible epitopes. Somegerm-line sequences have been suggested to induce t regulatory cells. Aprevious study with a therapeutic protein demonstrated a correlationbetween an immunologically active peptide, i.e. t-cell epitope, and theEpiMatrix prediction (Koren, E. et al. Clin Immunol 124, 26-32 (2007)).

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently features specified in onesection may be combined with features specified in other sections, asappropriate.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

TABLE 1 CDR sequence, affinity and stability characteristics ofparental, grafted and ABS-derived lead clones. % germ- line # FW inmuta- CDRL1* CDRL2 CDRL3 CDRH1 CDRH2 CDRH3 CDRs tions DPK9/DP54RASQSISSYLN AASSLQS QQSYSTPLT GFTFSSYWMS ANIKQDGSEKYYVDSVKG n/a 100 n/agermlines CL-Hum-RAGE RASQDVGIYVN RATNLAD LEFDEHPLT GFTFSNYWMTASIDNSGDNTYYPDSVKD GGDITTGFDY 45 5 Graft-RAGE RASQDVGIYVN RATNLADLEFDEHPLT GFTFSNYWMT ASIDNSGDNTYYPDSVKD GGDITTGFDY 45 0 C7-ABS-RAGERASQSIGSYLN RASSLAS LEFDEHPLT GFTFSSYWMS ASIDQDGSNKYYPDSVKG GGDITTGLDY74 0 DPK9/DP47 RASQSISSYLN AASSLQS QQSYS--TPLT GFTFSSYAMSSAISGSGGSTYYADSVKG n/a 100 n/a germlines Chim-A33 LASEFLFNGVS GASNLESLGGYSGSSGLT GIDFSHYGIS AYIYPNYGSVDYASWVNG DRGYYSGSRGTRLDL 39 n/aGraft-A33 LASEFLFNGVS GASNLES LGGYSGSSGLT GIDFSHYGIS SYIYPNYGSVDYASWVNGDRGYYSGSRGTRLDL 39 0 C6-ABS-A33 RASQFLFNGVS AASNLES QGGYSGSTGLTGFTFSHYGIS SYIYPSYGSTDYASSVKG DRGYYSGSRGTRLDL 56 0 DPL16/DP47QGDSLRSYYAS GKNNRPS NSRDSSGNHVV GFTFSSYAMS SAISGSGGSTYYADSVKG n/a 100n/a germlines Chim-pTau SGSD--YDYG- WNDKRPS GAYDGSAGGGI GFTLSSYQMMAGITSRGGVTGYGSAVKG PALDSDQCGFPEAGCIDA 41 n/a Graft-ABS- SGSD--YDYG-WNDKRPS GAYDGSAGGGI GFTLSSYQMM AGITSRGGVTGYGSAVKG PALDSDQCGFPEAGCIDA 410 pTau C21-ABS- QGDD--SYYG- GNDNRPS GAYDSSGGGGI GFTLSSYQMMAGITGRGGVTGYADSVKG PALDSDQCGFPEAGCIDA 65 1 pTau Com-ABS- QGDD--SYYG-GNNNRPS GSYDSSGGHGV GFTFSSYQMS SGITGRGGVTGYADSVKG PALDSDQCGFPEMGCIDA 761 pTau IC50 kD (nM) (nM) Tm HTRF SPR % Agg 60° C. (° C.) % Agg pH shockDPK9/DP54 germlines n/a n/a n/a n/a n/a CL-Hum-RAGE 10.3 31.0 14 77 3.2Graft-RAGE >61.9 ND 0 84 1.8 C7-ABS-RAGE 5.8 17.0 0 85 2.1 DPK9/DP47germlines n/a n/a n/a n/a n/a Chim-A33 2.9 2.1 0 74 1.5 Graft-A33 ND NDND ND ND C6-ABS-A33 2.7 2.1 0 74 0.4 DPL16/DP47 n/a n/a n/a n/a n/agermlines Chim-pTau 1.6 0.41 17 70 1.2 Graft-ABS-pTau >64.7 NB 90 66 0.8C21-ABS-pTau 2.1 0.25 3 70 1.2 Com-ABS-pTau ND 0.50 2 71 1.4 *The pTauCDR-L1 is shorter than its DPL16 counterpart by 3 amino acids. Sequencedashes in this CDR are added to show the spacing of sampled residues.Residues differing from human germline are underlined.

TABLE 2 Exemplary Human V_(H) germline sequences Human VH1 germlinesequence (from top to bottom, SEQ ID NOs. 56-69). VH1 FW1 CDR1 FW2 CDR2FW3 IGHV1-2 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYYMH.. WVRQAPGQGLEWMGRINP..NSGGTNYAQKFQG RVTSTRDTSISTAYMELS RLRSDDTVVYYCAR. IGHV1-3QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYAMH.. WVRQAPGQRLEWMGWINA..GNGNTKYSQKFQG RVTITRDTSASTAYMELS SLRSEDIAVYYCAR. IGHV1-8QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYDIN.. WVRQATGQGLEWMGWMNP..NSGNTGYAQKFQG RVTMTRNTSTSTAYMELS SLRSEDTAVYYCAR. IGHV1-18QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYGIS.. WVRQAPGQGLEWMGWISA..YNGNTNYAQKLOG RVTMTTDTSTSTAYMELR SLRSDDTAVYYCAR. IGHV1-24QVQLVQSGAEVKKPGASVKVSCKVS GYTLTELSMH.. WVRQAPGKGLEWMGGFDP..EDGETIYAQKFOG RVTMTEDTSTDTAYMELS SLRSEDTAVYYCAT. IGHV1-38-4QVQLVQSWAEVRKSGASVKVSCSFS GFTITSYGIH.. WVQQSPGQGLEWMGWINP..GNGSPSYAKKFQG RFTMTRDMSTITAYTDLS SLTSEDMAVYYYAR. IGHV1-45QMQLVQSGAEVKKTGSSVKVSCKAS GYTFTYRYLH.. WVRQAPGQALEWMGWITP..FNGNTNYAQKFQD RVTITRDRSMSTAYMELS SLRSEDTAMYYCAR. IGHV1-46QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYYMH.. WVRQAPGQALEWMGIINP..SGGSTSYAQKFQG RVTMTRDISTSTVYMELS SLRSEDTAVYYCAR. IGHV1-58QMQLVQSGPEVKKPGTSVKVSCKAS GFTFTSSAVQ.. WVRQARGQRLEWIGWIVV..GSGNTNYAQKFQE RVTITRDMSTSTAYMELS SLRSEDTAVYYCAA. IGHV1-68QVQLGQSEAEVKKPGASVKVSCKAS GYTFTCCSLH.. WLQQAPGQGLERMRWITL..YNGNTNYAKKFQG RVTITRDMSLRTAYIELS SLRSEDSAVYYWAR. IGHV1-68-2EVQLVQSGAEVKKPGATVKISCKVS GYTFTDYYMH.. WVQQAPGKGLEWMGLVDP..EDGETIYAEKFQG RVTITADTSTDTAYMELS SLRSEDTAVYYCAT. IGHV1-69QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYAIS.. WVRQAPGQGLEWMGGIIP..IFGTANYAQKFQG RVTITADESTSTAYMELS SLRSEDTAVYYCAR. IGHV1-69DQVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYAIS.. WVRQAPGQGLEWMGGIIP..IFGTANYAQKFQG RVTITADESTSTAYMELS SLRSEDTAVYYCAR. IGHV1-NL1QVQLLQPGVQVKKPGSSVKVSC*AS RYTFTKYFTR.. WV*QSPGQGHXWMG*INP..YNDNTHYAQTFWG RVTITSDRSMSTAYMELS XLRSEDMVVYYCVR. Human VH2germline sequence (from top to bottom, SEQ ID NOs. 70-73). VH2 FW1 CDR1FW2 CDR2 FW3 IGHV2-5 QITLKESGPTLVKPTQTLTLTCTFS GFSLSTSGVGVGWIRQPPGKALEWL ALIY...WNDDKRYSPSLKS RLTITKDISKNQVVLTMT NMDFVDTATYYCAHRIGHV2-10 QVTLKESGPALVKPTQTLMLTCTFS GFSLSTSGMGVG *ICQPSAKALEWLAHIY...*NDNKYYSPSLKS RLIISKDTSKNEVVLTVI NMDIVDTATHYCARR IGHV2-26QVTLKESGPVLVKPTETLTLTCTVS GFSLSNARMGVG WIRQPPGKALEWLAHIF...SNDEKSYSTSLKS RLTISKDTSKSQVVLTMT NMDPVDTATYYCARI IGHV2-70QVTLRESGPALVKPTQTLTLTCTFS GFSLSTSGMCVS WIRQPPGKALEWLALID...WDDSKYYSTSLKT RLTISKDTSKNQVVLTMT NMDPVDTATYYCARI Human VH3germline sequence (from top to bottom, SEQ ID NOs: 74-114): VH3 FW1 CDR1FW2 CDR2 FW3 IGHV3-7 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYWMS..WVRQAPGKGLEWV ANIKQ..DGSEKYYVDSVKG RFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR.IGHV3-9 EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYAMH.. WVRQAPGKGLEWVSGISN..NSGSIGYADSVKG RFTISRDNAKNSLYLQMN SLRAEDTALYYCAKD IGHV3-11QVQLVESGGGLVKPGGSLRLSCAAS GFTFSDYYMS.. WIRQAPGKGLEWVSYISS..SGSTIYYADSVKG RFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR. IGHV3-13EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYDMH.. WVRQAIGKGLEWVSAIG...TAGDTYYPGSVKG RFTISRENAKNSLYLQMN SLRAGDTAVYYCAR. IGHV3-15EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAWMS.. WVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMN SLKTEDTAVYYCTT. IGHV3-16EVQLVESGGGLVQPGGSLRLSCAAS GFTFSNSDMN.. WARKAPGKGLEWVSGVSW..NGSRTHYVDSVKR RFTISRDNSRNSLYLQKN RRRAEDMAVYYCVR. IGHV3-19TVQLVESGGGLVEPGGSLRLSCAAS GFTFSNSDMN.. WVRQAPGKGLEWVSGVSW..NGSRTHYADSVKG RFTISRDNSRNFYLQQMN SLRPEDMAVYYCVR. IGHV3-20EVQLVESGGGLVRPGGSLRLSCAAS GFTFDDYGMS.. WVRQAPGKGLEWVSGINW..NGGSTGYADSVKG RFTISRDNAKNSLYLQMN SLRAEDTALYHCAR. IGHV3-21EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN.. WVRQAPGKGLEWVSSISS..SSSYIYYADSVKG RFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR. IGHV3-22EVHLVESGGALVQPGGSLRLSCAAS GFTFSYYYMS.. GVRQAPGKGLEWVGFIRNKANGGTTE*TTSVKG RFTISRDDSKSITYLQMK SLKTEDTAVYYCAR. IGHV3-23EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS.. WVRQAPGKGLEWVSAISG..SGGSTYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAK. IGHV3-23DEVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS.. WVRQAPGKGLEWVSAISG..SGGSTYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAK. IGHV3-25EMQLVESGGGLQKPAWSPRLSCAAS QFTFSSYYMN.. CVRQAPGNGLELV*QVNP..NGGSTYLIDSGKD RFNTSRDNARNTLHLQMN SLKTEDTALY*CTR. IGHV3-29EVELIEPTEDLRQPGKFLRLSCVAS RFAFSSF*MS.. PVHQSAGKGLE*VIDIKD..DGSQIHHADSVKG RFSISKDNAKNSLYLQMN SQRTEDMAVYGCT*G IGHV3-30-2EVQLVESGEDPRQPGGSLRLSCADS GLTFSSY*RN.. SVSQAPGKGLE*VVDIQC..DGSQICYA*SLKS KFTISKENAKNSLYLLMN SLRAAGTAVCYCM*G IGHV3-30-3QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYAMH.. WVRQAPGKGLEWVAVISY..DGSNKYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-30QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYAMH.. WVRQAPGKGLEWVAVISY..DGSNKYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-32EVELIESIEDLRQPGKFLRLSCVAS RFAFSSF*MS.. RVHQSPGKGLE*VIDIKD..DGSQIHHADSVKG RFSISKDNAKNSLYLQMN TQRAEDVAVYGYT*G IGHV3-33-2EVQLVESGEDPRQPGGSLRLSCADS GLTFSSY*MS.. SVSQAPGKGLE*VVDIQC..DGSQICYAQSVKG KFTISKENAKNSLYLQMN SLRAEGTAVCYCM*G IGHV3-33QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMH.. WVRQAPGKGLEWVAVIWY..DGSNKYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-35EVQLVESGGGLVQPGGSLRLSCAAS GFTFSNSDMN.. WVHQAPGKGLEWVSGVSW..NGSRTHYADSVKG RFTISRDNSRNTLYLQTN SLRAEDTAVYYCVR. IGHV3-38-3EVQLVESRGVLVQPGGSLRLSCAAS GFTVSSNEMS.. WVRQAPGKGLEWVSSIS....GGSTYYADSRKG RFTISRDNSKNTLHLQMN SLRAEDTAVYYCKK. IGHV3-38EVQLVESGGGLVQPRGSLRLSCAAS GFTVSSNEMS.. WIRQAPGKGLEWVSSIS....GGSTYYADSRKG RFTISRDNSKNTLYLQMN NLRAEGTAAYYCARY IGHV3-43EVQLVESGGVVVQPGGSLRLSCAAS GFTFDDYTMH.. WVRQAPGKGLEWVSLISW..DGGSTYYADSVKG RFTISRDNSKNSLYLQMN SLRTEDTALYYCAKD IGHV3-43DEVQLVESGGVVVQPGGSLRLSCAAS GFTFDDYAMH.. WVRQAPGKGLEWVSLISW..DGGSTYYADSVKG RFTISRDNSKNSLYLQMN SLRAEDTALYYCAKD IGHV3-47EVQLVESGGGLVQPGGSLRPSCAAS GFAFSSYALH.. WVRRAPGKGLEWVSAIG...TGGDTYYADSVMG RFTISRDNAKKSLYLHMN SLIAEDMAVYYCAR. IGHV3-48EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYSMN.. WVRQAPGKGLEWVSYISS..SSSTIYYADSVKG RFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR. IGHV3-49EVQLVESGGGLVQPGGSLRLSCTAS GFTFGDYAMS.. WFRQAPGKGLEWVGFIRSKAYGGTTEYTASVKG RFTISRDGSKSIAYLQMN SLKTEDTAVYYCTR. IGHV3-52EVQLVESG*GLVQPGGSLRLSCAAS GFTFSSSWMH.. WVCQAPEKGLEWVADIKC..DGSEKYYVDSVKG RLTISRDNAKNSLYLQVN SLRAEDMTVYYCVR. IGHV3-53EVQLVESGGGLIQPGGSLRLSCAAS GFTVSSNYMS.. WVRQAPGKGLEWVSVIY...SGGSTYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-54EVQLVESERNQRQLGGSLRLSCADS GLTFSSY*MS.. SDSQAPGKGLE*VVDI**..DRSQLCYAQSVKS RFTISKENAKNSLCLQMN SLRAEGTAVYYCM*. IGHV3-62EVQLVESGEGLVQPGGSLRLSCAAS GFTFSSSAMH.. WVRQAPRKGL*WVSVIST..SGDTVLYTDSVKG RFTISRDNAQNSLSLQMN SLRAEGTVVYYCVK. IGHV3-63EVELIESIEGLRQLGKFLRLSCVAS GFTFSSY*MS.. WVNETLGKGLEGVIDVKY..DGSQIYHADSVKG RFTISKDNAKNSPYLQVN SLRAEDMTMHGCT*G IGHV3-64EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYAMH.. WVRQAPGKGLEYVSAISS..NGGSTYYANSVKG RFTISRDNSKNTLYLQMG SLRAEDMAVYYCAR. IGHV3-65EVQLVESGGGLVQPGGSLRLSCAAS GFTVSSNYMS.. WVRQAPGKGLEWVSVIY...SGGSTYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-69-1EVQLVESGGGLVKPGGSLRLSCAAS GFTFSDYYMN.. WVRQAPGKGLEWV SSIS..SSSTIYYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCAR. IGHV3-71 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMS.. WVRQAPGKGLEWV GFIRNKANGGTTE*TTSVKG RFTISRDDSKSITYLQMNSLRAEDTAVYYCAR. IGHV3-72 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSDHYMD..WVRQAPGKGLEWV GRIRNKANSYTTEYAASVKG RFTISRDDSKNSLYLQMN SLKTEDTAVYYCAR.IGHV3-73 EVQLVESGGGLVQPGGSLKLSCAAS GFTFSGSAMH.. WVRQASGKGLEWVGRIRSKANSYATAYAASVKG RFTISRDDSKNTAYLQMN SLKTEDTAVYYCTR. IGHV3-74EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYWMH.. WVRQAPGKGLVWVSRINS..DGSSTSYADSVKG RFTISRDNAKNTLYLQMN SLRAEDTAVYYCAR. IGHV3-NL1QVQLVESGGGVVQPGGSLRLSCAAS GFTFSSYGMH.. WVRQAPGKGLEWVEVIYS..GGSSTYYADSVKG RFTISRDNSKNTLYLQMN SLRAEDTAVYYCAK. Human VH4germline sequence (from top to bottom, SEQ ID NOs. 115-125): VH4 FW1CDR1 FW2 CDR2 FW3 IGHV4-4 QVQLQESGPGLVKPPGTLSLTCAVS GGSISSSNWWS.WVRQPPGKGLEWI GEIY...HSGSTNYNPSLKS RVTISVDKSKNQFSLKLS SVTAADTAVYCCAR.IGHV4-28 QVQLQESGPGLVKPPDTLSLTCAVS GYSISSSNWWG. WIRQPPGKGLEWIGYIY...YSGSTYYNPSLKS RVTMSVDTSKNQFSLKLS SVTAVDTAVYYCAR. IGHV4-30-2QLQLQESGSGLVKPSQTLSLTCAVS GGSISSGGYSWS WIRQPPGKGLEWIGYIY...HSGSTYYNPSLKS RVTISVDRSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-30-4QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGDYYWS WIRQPPGKGLEWIGYIY...YSGSTYYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-31QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGGYYWS WIRQHPGKGLEWIGYIY...YSGSTYYNPSLKS LVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-34QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYYWS.. WIRQPPGKGLEWIGEIN...HSGSTNYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-38-2QVQLQESGPGLVKPSETLSLTCAVS GYSISSGYYWG. WIRQPPGKGLEWIGSIY...HSGSTYYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-39QLQLQESGPGLVKPSETLSLTCTVS GGSISSSSYYWG WIRQPPGKGLEWIGSIY...YSGSTYYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-55QVQLQESGPGLVKPSETLSLTCAVS GDSISSGNW*I. WVRQPPGKGLEWIGEIH...HSGSTYYNPSLKS RITMSVDTSKNQFYLKLS SVTAADTAVYYCAR. IGHV4-59QVQLQESGPGLVKPSETLSLTCTVS GGSISSYYWS.. WIRQPPGKGLEWIGYIY...YSGSTNYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. IGHV4-61QVQLQESGPGLVKPSETLSLTCTVS GGSVSSGSYYWS WIRQPPGKGLEWIGYIY...YSGSTNYNPSLKS RVTISVDTSKNQFSLKLS SVTAADTAVYYCAR. Human VH5germline sequence (from top to bottom, SEQ ID NOs. 126-128): VH5 FW1CDR1 FW2 CDR2 FW3 IGHV5-10-1 EVQLVQSGAEVKKPGESLRISCKGS GYSFTSYWIS..WVRQMPGKGLEWM GRIDP..SDSYTNYSPSFQG HVTISADKSISTAYLQWS SLKASDTAMYYCAR.IGHV5-51 EVQLVQSGAEVKKPGESLKISCKGS GYSFTSYWIG.. WVRQMPGKGLEWMGIIYP..GDSDTRYSPSFQG QVTISADKSISTAYLQWS SLKASDTAMYYCAR. IGHV5-78EVQLLQSAAEVKRPGESLRISCKIS GYSFTSYWIH.. WVRQMPGKGKEWMGSIYP..GNSDTRYSPSFQG HVTISADSSSSTAYLQWS SLKASDAAMYYCVR. Human VH6germline sequence (SEQ ID NO: 129) VH6 FW1 CDR1 FW2 CDR2 FW3 IGHV6-1QVQLQQSGPGLVKPSQTLSLTCAIS GDSVSSNSAAWN WIRQSPSRGLEWLGRIYY.RSKWYNDYAVSVKS RITINPDTSKNQFSLQLN SVTPEDTAVYYCAR. Human VH7germline sequence (from top to bottom, SEQ ID NOs. 130-132): VH7 FW1CDR1 FW2 CDR2 FW3 IGHV7-4-1 QVQLVQSGSELKKPGASVKVSCKAS GYTFTSYAMN..WVRQAPGQGLEWM GWINT..NTGNPTYAQGFTG RFVFSLDTSVSTAYLQIC SLKAEDTAVYYCAR.IGHV7-34-1 -LQLVQSGPEVKKPGASVKVSYKSS GYTFIIYGMN.. WV**IPGQGFEWM*WIIT..YTGNPTYTHGFTG WFVFSMDTSVSTACLQIS SLKAEDTAEYYCAR. IGHV7-81QVQLVQSGHEVKQPGASVKVSCKAS GYSFTTYGMN.. WVPQAPGQGLEWMGWFNT..YTGNPTYAQGFTG RFVFSMDTSASTAYLQIS SLKAEDMAMYYCAR.

TABLE 3 Exemplary Human Vκ germline sequencesHuman VK1 germline sequence (from top to bottom, SEQ ID NOs. 133-145):VK1 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV1-5 DIQMTQSPSTLSASVGDRVTITCRASQ......SISSWLA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEF QQYNSYSTLTISSLQPDDFATYYC IGKV1-6 AIQMTQSPSSLSASVGDRVTITC RASQ......GIRNDLGWYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDF LQDYNYP TLTISSLQPEDFATYYCIGKV1-8 AIRMTQSPSSFSASTGDRVTITC RASQ......GISSYLA WYQQKPGKAPKLLIYAASTLQS GVPSRFSGSGSGTDF QQYYSYP ILTISCLQSEDFATYYC IGKV1-9DIQLTQSPSFLSASVGDRVTITC RASQ......GISSYLA WYQQKPGKAPKLLIY AASTLQSGVPSRFSGSGSGTEF QQLNSYP TLTISSLQPEDFATYYC IGKV1-12DIQMTQSPSSVSASVGDRVTITC RASQ......GISSWLA WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTD QQANSFP FTLTISSLQPEDFATYYC IGKV1-13AIQLIQSPSSLSASVGDRVTITC RASQ......GISSALA *YQQKPGKAPKLLIY DASSLESGVPSRFSGSGSGTDF QQFNNYP ILTISSLQPEDFATYYC IGKV1-16DIQMTQSPSSLSASVGDRVTITC RASQ......GISNYLA WFQQKPGKAPKSLIY AASSLQSGVPSRFSGSGSGTDF QQYNSYP TLTISSLQPEDFATYYC IGKV1-17DIQMTQSPSSLSASVGDRVTITC RASQ......GIRNDLG WYQQKPGKAPKRLIY AASSLQSGVPSRFSGSGSGTEF LQHNSYP TLTISSLQPEDFATYYC IGKV1-27DIQMTQSPSSLSASVGDRVTITC RASQ......GISNYLA WYQQKPGKVPKLLIY AASTLQSGVPSRFSGSGSGTDF QKYNSAP TLTISSLQPEDVATYYC IGKV1-33DIQMTQSPSSLSASVGDRVTITC QASQ......DISNYLN WYQQKPGKAPKLLIY DASNLETGVPSRFSGSGSGTDF QQYDNLP TFTISSLQPEDIATYYC IGKV1-37DIQLTQSPSSLSASVGDRVTITC RVSQ......GISSYLN WYRQKPGKVPKLLIY SASNLQSGVPSRFSGSGSGTDF QRTYNAP TLTISSLQPEDVATYYG IGKV1-39DIQMTQSPSSLSASVGDRVTITC RASQ......SISSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDF QQSYSTP TLTISSLQPEDFATYYC IGKV1-NL1DIQMTQSPSSLSASVGDRVTITC RASQ......GISNSLA WYQQKPGKAPKLLLY AASRLESGVPSRFSGSGSGTD QQYYSTP YTLTISSLQPEDFATYYCHuman VK1D germline sequence (from top to bottom, SEQ ID NOs. 146-155):VK1D FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV1D-8 VIWMTQSPSLLSASTGDRVTISCRMSQ......GISSYLA WYQQKPGKAPELLIY AASTLQS GVPSRFSGSGSGTD QQYYSFPFTLTISCLQSEDFATYYC IGKV1D-12 DIQMTQSPSSVSASVGDRVTITC RASQ......GISSWLAWYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTD QQANSFP FTLTISSLQPEDFATYYCIGKV1D-13 AIQLTQSPSSLSASVGDRVTITC RASQ......GISSALA WYQQKPGKAPKLLIYDASSLES GVPSRFSGSGSGTD QQFNNYP FTLTISSLQPEDFATYYC IGKV1D-16DIQMTQSPSSLSASVGDRVTITC RASQ......GISSWLA WYQQKPEKAPKSLIY AASSLQSGVPSRFSGSGSGTD QQYNSYP FTLTISSLQPEDFATYYC IGKV1D-17NIQMTQSPSAMSASVGDRVTITC RARQ......GISNYLA WFQQKPGKVPKHLIY AASSLQSGVPSRFSGSGSGTE LQHNSYP FTLTISSLQPEDFATYYC IGKV1D-33DIQMTQSPSSLSASVGDRVTITC QASQ......DISNYLN WYQQKPGKAPKLLIY DASNLETGVPSRFSGSGSGTD QQYDNLP FTFTISSLQPEDIATYYC IGKV1D-37DIQLTQSPSSLSASVGDRVTITC RVSQ......GISSYLN WYRQKPGKVPKLLIY SASNLQSGVPSRFSGSGSGTD QRTYNAP FTLTISSLQPEDVATYYG IGKV1D-39DIQMTQSPSSLSASVGDRVTITC RASQ......SISSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTD QQSYSTP FTLTISSLQPEDFATYYC IGKV1D-42DIQMIQSPSFLSASVGDRVSIIC WASE......GISSNLA WYLQKPGKSPKLFLY DAKDLHPGVSSRFSGRGSGTDF KQDFSYP TLTIISSLKPEDFAAYYC IGKV1D-43AIRMTQSPFSLSASVGDRVTITC WASQ......GISSYLA WYQQKPAKAPKLFIY YASSLQSGVPSRFSGSGSGTD QQYYSTP YTLTISSLQPEDFATYYCHuman VK2 germline sequence (from top to bottom, SEQ ID NOs. 156-162):VK2 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV2-4 DIVMTQHLLSLPIPLGEPASISCRSSQSLLHS.DGNTYLD WYLQKPGQSPQLLIY TISNKFY GVPNKFSGSRSGTGF EQGLQGPTLKFSKVEAEDVGVYCC IGKV2-18 DIVMTQTPPSLPVNPGEPASISC RSSQSLLHS.NGYTYLHWYLQKPGQSPQLLIY RVSNHLS GVPDRFSGSGSGSDF MQATQFP TLKISWVEAEDVGVYYCIGKV2-24 DIVMTQTPLSSPVTLGQPASISC RSSQSLVHS.DGNTYLS WLQQRPGQPPRLLIVKISNRFS GVPDRFSGSGAGTDF MQATQFP TLKISRVEAEDVGVYYC IGKV2-28DIVMTQSPLSLPVTPGEPASISC RSSQSLLHS.NGYNYLD WYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDF MQALQTP TLKISRVEAEDVGVYYC IGKV2-29DIVMTQSPLSLSVTPGQPASISC RSSQSLLHS.DGKTYLY WYLQKPGQSPQLLIY EVSSRFSGVPDRFSGSGSGTDF MQGIHLP TLKISRVEAEDVGVYY* IGKV2-30DVVMTQSPLSLPVTLGQPASISC RSSQSLVYS.DGNTYLN WFQQRPGQSPRRLIY KVSNRDSGVPDRFSGSGSGTD MQGTHWP FTLKISRVEAEDVGVYYC IGKV2-40DIVMTQTPLSLPVTPGEPASISC RSSQSLLDSDDGNTYLD WYLQKPGQSPQLLIY TLSYRASGVPDRFSGSGSGTDF MQRIEEP TLKISRVEAEDVGVYYCHuman VK2D germline sequence (from top to bottom, SEQ ID NOs. 163-169):VK2D FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV2D-18 DIVMTQTPPSLPVNPGEPASISCRSSQSLLHS.NGYTYLH WYPQKPGQSPQLLIY RVSSRFS GVPDRFSGSGSGSDF MQATQFPTLKISWVEAEDVGVYYC IGKV2D-24 DIVMTQTPLSSPVTLGQPASISF RSSQSLVHS.DGNTYLSWLQQRPGQPPRLLIY KVSNRFS GVPDRFSGSGAGTDFT TQATQFP LKISRVEAEDVGVYYCIGKV2D-26 EIVMTQTPLSLSITPGEQASISC RSSQSLLHS.DGYTYLY WFLQKARPVSTLLIYEVSNRFS GVPDRFSGSGSGTDFTL MQDAQDP KISRVEAEDFGVYYC IGKV2D-28DIVMTQSPLSLSVTPGEPASISC RSSQSLLHS.NGYNYLD WYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTD MQALQTP FTLKISRVEAEDVGVYYC IGKV2D-29DIVMTQSPLSLSVTPGQPASISC KSSQSLLHS.DGKTYLY WYLQKPGQPPQLLIY EVSNRFSGVPDRFSGSGSGTD MQSIQLP FTLKISRVEAEDVGVYYC IGKV2D-30DVVMTQSPLSLPVTLGQPASISC RSSQSLVYS.DGNTYLN WFQQRPGQSPRRLIY KVSNWDSGVPDRFSGSGSGTD MQGTHWP FTLKISRVEAEDVGVYYC IGKV2D-40DIVMTQTPLSLPVTPGEPASISC RSSQSLLDSDDGNTYLD WYLQKPGQSPQLLIY TLSYRASGVPDRFSGSGSGTDF MQRIEEP TLKISRVEAEDVGVYYCHuman VK3 germline sequence (from top to bottom, SEQ ID NOs. 170-178):VK3 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV3-7 EIVMTQSPPTLSLSPGERVTLSCRASQS.....VSSSYLT WYQQKPGQAPRLLIY GASTRAT SIPARFSGSGSGTDF QQDHNLPTLTISSLQPEDFAVYYC IGKV3-11 EIVLTQSPATLSLSPGERATLSC RASQ......SVSSYLAWYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDF QQRSNWP TLTISSLEPEDFAVYYCIGKV3-15 EIVMTQSPATLSLSPGERATLSC RASQ......SVSSNLA WYQQKPGQAPRLLIYGASTRAT GIPARFSGSGSGTEF QQYNNWP TLTISSLQSEDFAVYYC IGKV3-20EIVLTQSPATLSLSPGERATLSC RASQ......VSSSYLA WYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDF QQYGSSP TLTISRLEPEDFAVYYC IGKV3-NL1EIVLTQSPATLSLSPGERATLSC RASQ......SVSSYLA WYQQKPGQAPRLLIY GASTRATGIPARFSGSGSGTEF Q...... TLTISSLQSEDFAVYYC IGKV3-NL2EIVLTQSPATLSLSPGERATLSC RASQ......GVSSYLA WYQQKPGQAPRLLIY DASSRATGIPARFSGSGSGTDF Q...... TLTISSLEPEDFAVYYC IGKV3-NL3EIVLTQSPGTLSLSPGERATLSC RASQS.....VSSSYLA WYQQKPGLAPRLLIY GASTRATGIPARFSGSGSGTEF Q...... TLTISRLESEDFAVYYC IGKV3-NL4EIVLTQSPATLSLSPGERATLSC RASQ......GVSSNLA WYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDF QQRSNWH TLTISSLEPEDFAVYYC IGKV3-NL5EIVLTQSPATLSLSPGERATLSC RASQS.....VSSSYLA WYQQKPGQAPRLLIY DASSRATGIPDRFSGSGSGTDF QQRSNWH TLTISRLEPEDFAVYYCHuman VK3D germline sequence (from top to bottom, SEQ ID NOs. 179-182):VK3D FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV3D-7 EIVMTQSPATLSLSPGERATLSCRASQS.....VSSSYLS WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTDF QQDYNLPTLTISSLQPEDFAVYYC IGKV3D-11 EIVLTQSPATLSLSPGERATLSC RASQ.....GVSSYLAWYQQKPGQAPRLLIY DASNRAT GIPARFSGSGPGTDF QQRSNWH TLTISSLEPEDFAVYYCIGKV3D-15 EIVMTQSPATLSVSPGERATLSC RASQ.....SVSSNLA WYQQKPGQAPRLLIYGASTRAT GIPARFSGSGSGTDF QQYNNWP TLTISSLQSEDFAVYYC IGKV3D-20EIVLTQSPATLSLSPGERATLSC GASQS.....VSSSYLA WYQQKPGLAPRLLIY DASSRATGIPDRFSGSGSGTDF QQYGSSP TLTISSLEPEDFAVYYCHuman VK4 germline sequence (SEQ ID NO: 183) VK4 FW1 CDR1 FW2 CDR2 FW3CDR3 IGKV4-1 DIVMIQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDF QQYYSTP TLTISSLQAEDVAVYYCHuman VK5 germline sequence (SEQ ID NO: 184) VK5 FW1 CDR1 FW2 CDR2 FW3CDR3 IGKV5-2 ETTLTQSPAFMSATPGDKVNISC KASQ......DIDDDMN WYQQKPGEAAIFIIQEATTLVP GIPPRFSGSGYGTDF LQHDNFP TLTINNIESEDAAYYFCHuman VK6 germline sequence (SEQ ID NO: 185) VK6 FW1 CDR1 FW2 CDR2 FW3CDR3 IGKV6-21 EIVLIQSPDFQSVTPKEKVTIIC RASQ......SIGSSLH WYQQKPDQSPKLLIKYASQSFS GVPSRFSGSGSGTDF HQSSSLP TLTINSLEAEDAATYYCHuman VK6D germline sequence (from top to bottom, SEQ ID NOs. 186-187):VK6D FW1 CDR1 FW2 CDR2 FW3 CDR3 IGKV6D-21 EIVLTQSPDFQSVTPKEKVTITCRASQ......SIGSSLH WYQQKPDQSPKLLIK YASQSFS GVPSRFSGSGSGTDF HQSSSLPTLTINSLEAEDAATYYC IGKV6D-41 DVVMIQSPAFLSVTPGEKVTITC QASE......GIGNYLYWYQQKPDQAPKLLIK YASQSIS GVPSRFSGSGSGTDFTF QQGNKHP TISSLEAEDAATYYCHuman VK7 germline sequence (SEQ ID NO: 188) VK7 FW1 CDR1 FW2 CDR2 FW3CDR3 IGKV7-3 DIVLTQSPASLAVSPGQRATITC RASESVSF..LGINLIH WYQQKPGQPPKLLIYQASNKDT GVPARFSGSGSGTDF LQSKNFP TLTINPVEANDTANYYC

TABLE 4 Exemplary Human V_(λ) germline sequencesHuman Vλ1 germline sequences (from top to bottom, SEQ ID NOs. 189-196):Vλ1 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV1-36 QSVLTQPPSVS SGSSSN..IGNNAVNWYQQLPGKAPKLLIY YDDLL.....PS GVSDRFSGSK..SGTSAS AAWDDSLNG... EAPRQRVTISCLAISGLQSEDEADYYC IGLV1-40 QSVLTQPPSVS TGSSSNI.GAGYDVH WYQQLPGTAPKLLIYGNSNR.....PS GVPDRFSGSK..SGTSAS QSYDSSLSG... GAPGQRVTISCLAITGLQAEDEADYYC IGLV1-41 QSVLTQPPSVS SGSSSD..MGNYAVS WYQQLPGTAPKLLIYENNKR.....PS GIPDRFSGSK..SGTSAT LAWDTSPRA... AAPGQKVTISCLGITGLWPEDEADYYC IGLV1-44 QSVLTQPPSAS SGSSSN..IGSNTVN WYQQLPGTAPKLLIYSNNQR.....PS GVPDRFSGSK..SGTSAS AAWDDSLNG... GTPGQRVTISCLAISGLQSEDEADYYC IGLV1-47 QSVLTQPPSAS SGSSSN..IGSNYVY WYQQLPGTAPKLLIYRNNQR.....PS GVPDRFSGSK..SGTSAS AAWDDSLSG... GTPGQRVTISCLAISGLRSEDEADYYC IGLV1-50 QSVLTQPPSVS TGSSSNI.GAGYVVH WYQQLPGTAPKLLIYGNSNR.....PS GVPDQFSGSK..SGTSAS KAWDNSLNA... GAPGQRVTISCLAITGLQSEDEADYYC IGLV1-51 QSVLTQPPSVS SGSSSN..IGNNYVS WYQQLPGTAPKLLIYDNNKR.....PS GIPDRFSGSK..SGTSAT GTWDSSLSA... AAPGQKVTISCLGTTGLQTGDEADYYC IGLV1-52 QSVLTQPPSVS TGSSSNTGTGYNVNC WQ*LPRTDPKLLPHGDKNW.....PS WVSDQFSGSK..SGSLAS QSRDIC*VL... WATRQRLTVSCLGTTGIWAEDKTDYHCHuman Vλ2 germline sequences (from top to bottom, SEQ ID NOs. 197-205):Vλ2 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV2-5 QSALTQPPSVS TGTSSDV.GSYDYVSWYQQHPGTVPKPMIY NVNTQ.....PS GVPDRFSGSK..SGNTAS CSYTSSAT*... GSPGQSVTISCMTISGLQAEDEADY*C IGLV2-8 QSALTQPPSAS TGTSSDV.GGYNYVS WYQQHPGKAPKLMIYEVSKR.....PS GVPDRFSGSK..SGNTAS SSYAGSNNF... GSPGQSVTISCLTVSGLQAEDEADYYC IGLV2-11 QSALTQPRSVS TGTSSDV.GGYNYVS WYQQHPGKAPKLMIYDVSKR.....PS GVPDRFSGSK..SGNTAS CSYAGSYTF... GSPGQSVTISCLTISGLQAEDEADYYC IGLV2-14 QSALTQPASVS TGTSSDV.GGYNYVS WYQQHPGKAPKLMIYEVSNR.....PS GVSNRFSGSK..SGNTAS SSYTSSSTL... GSPGQSITISCLTISGLQAEDEADYYC IGLV2-18 QSALTQPPSVS TGTSSDV.GSYNRVS WYQQPPGTAPKLMIYEVSNR.....PS GVPDRFSGSK..SGNTAS SLYTSSSTF... GSPGQSVTISCLTISGLQAEDEADYYC IGLV2-23 QSALTQPASVS TGTSSDV.GSYNLVS WYQQHPGKAPKLMIYEGSKR.....PS GVSNRFSGSK..SGNTAS CSYAGSSTL... GSPGQSITISCLTISGLQAEDEADYYC IGLV2-33 QSALTQPPFVS TGTSSDV.GDYDHVF WYQKRLSTTSRLLIYNVNTR.....PS GISDLFSGSK..SGIMAS SLYSSSYTF... GAPGQSVTISCLTISGLKSEVEANYHC IGLV2-34 QSVLTQPRSVS TGTSSDI.GGYDLVS WOQ*HPGKAPKLMIYDVANW.....PS GAPGCFSGSK..SGNTAS SSYAGSYNF... RSPGQ*VTIFCLTISGLQAEDEADYYC IGLV2-NL1 QSVLTQPRSVS TGTSSDI.GGYDLVS WOQ*HPGKAPKLMIYDVGNW.....PS GAPGCFSGSK..SGNTAS SSYAGSYNF... RSPGQ*VTIFCLTISGLQAEDEADYYCHuman Vλ3 germline sequences (from top to bottom, SEQ ID NOs. 206-218):Vλ3 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV3-1 SYELTQPPSVS SGDK....LGDKYACWYQQKPGQSPVLVTY QDSKR.....PS GIPERFSGSN..SGNTAT QAWDSSTA.... VSPGQTASITCLTISGTQAMDEADYYC IGLV3-9 SYELTQPLSVS GGNN....IGSKNVH WYQQKPGQAPVLVTYPDSNR.....PS GIPERFSGSN..SGNTAT QVWDSSTA.... VALGQTARITCLTISRAQAGDEADYYC IGLV3-10 SYELTQPPSVS SGDA....LPKKYAY WYQQKSGQAPVLVTYEDSKR.....PS GIPERFSGSS..SGTMAT YSTDSSGNH... VSPGQTARITCLTISGAQVEDEADYYC IGLV3-12 SYELTQPHSVS GGNN....IGSYAVH WYQQKPGQDPVLVTYSDSNR.....PS GIPERFSGSN..PGNTTT QVWDSSSDH... VATAQMARITCLTISRTEAGDEADYYC IGLV3-13 SYELTQPPAVS SGDV....LRDNYAD WYPQKPGQAPVLVTYKDGER.....PS GIPERFSGST..SGNTTA FSGD*NN..... VSPGQTARISCLTISRVLTKGGADYYC IGLV3-15 SYELTQPPSVS SGEA....LPKKYAY WYQQKPGQFPVLVTYKDSER.....PS GIPERFSGSS..SGTIVT LSADSSGTY... VSLGQMARITCLTISGVQAEDEADYYC IGLV3-19 SSELTQDPAVS QGDS....LRSYYAS WYQQKPGQAPVLVTYGKNNR.....PS GIPDRFSGSS..SGNTAS NPRDSSGNH... VALGQTVRITCLTITGAQAEDEADYYC IGLV3-21 SYVLTQPPSVS GGNN....IGSKSVH WYQQKPGQAPVLVTYYDSDR.....PS GIPERFSGSN..SGNTAT QVWDSSSDH... VAPGKTARITCLTISRVEAGDEADYYC IGLV3-22 SYELTQLPSVS SGDV....LGENYAD WYQQKPGQAPELVTYEDSER.....YP GIPERFSGST..SGNTTT LSGDEDN..... VSPGQTARITCLTISRVLTEDEADYYC IGLV3-25 SYELNQPPSVS SGDA....LPKQYAY WYQQKPGQAPVLVTYKDSER.....PS GIPERFSGSS..SGTTVT QSADSSGTY... VSPGQTARITCLTISGVQAEDEADYYC IGLV3-27 SYELTQPSSVS SGDV....LAKEYAR WFQQKPGQAPVLVTYKDSER.....PS GIPERFSGSS..SGTTVT YSAADNN..... VSPGQTARITCLTISGAQVEDEADYYC IGLV3-31 SSELSQEPAVS QGDS....IEDSVVN WYKQKPSQAPGLVT*INSVQ.....SS GIPKKFSGSS..SGNMAT QSWDSSRTH... VALG*TARITCLTITGTQVEDKADYYC IGLV3-32 SSGPTQVPAVS QGDS....MEGSYEH WYQQKPGQAPVLVTYDSSDR.....PS GIPERFSGSE..SGNTTT QLIDNHA..... VALGQMARITCLTITGAQAEDEADYYTHuman Vλ4 germline sequences (from top to bottom, SEQ ID NOs. 219-221):Vλ4 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV4-3 LPVLTQPPSAS TLSSE...HSTYTIEWYQQRPGRSPQYIMK VKSDGSHSK.GD GIPDRFMGSS..SGADRY GESHTTDGQVG* ALLGASIKLTCLTFSNLQSDDEAEYHC IGLV4-50 QPVLTQSSSAS TLSSG...HSSYIIA WHQQQPGKAPRYLMKLEGSGSYNK.GS GVPDRFSGSS..SGADRY ETWDSNT..... ASLGSSVKLTCLTISNLQLEDEADYYC IGLV4-59 QPVLTQSPSAS TLSSG...HSSYAIA WHQQQPEKGPRYLMKLNSDGSHSK.GS GIPDRFSGSS..SGAERY QTWGTGI..... ASLGASVKLTCLTISSLQSEDEADYYCHuman Vλ5 germline sequences (from top to bottom, SEQ ID NOs. 222-2261):Vλ5 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV5-37 QPVLTQPPSSSA TLPSDIN.VGSYNIYWYQQKPGSPPRYLLY YYSDSDKGQ.GS GVPSRFSGSKDASANTGI MIWPSNAS.... SPGESARLTCLLISGLQSEDERDYYC IGLV5-39 QPVLTQPTSLSA TLRSDIN.VGTYRIY WYQQKPGSLPRYLLRYKSDSDKQQ.GS GVPSRFSGSKDASTNAG AIWYSSTS.... SPGASARFTC LLLISGLQSEDEADYYCIGLV5-45 QAVLTQPASLSA TLRSGIN.VGTYRIY WYQQKPGSPPQYLLR YKSDSDKQQ.GSGVPSRFSGSKDASANAGI MIWHSSAS.... SPGASASLTC LLISGLQSEDEADYYC IGLV5-48QPVLTQPTSLSA TLRSGIN.LGSYRIF WYQQKPESPPRYLLS YYSDSSKRQ.GSGVPSRFSGSKDASSNAGI MIWHSSAS.... SPGASARLTC LVISGLQSEDEADYYC IGLV5-52QPVLTQPSSHSA MLSSGFS.VGDFWIR WYQQKPGNPPRYLLY YHSDSNKGQ.GSGVPSRFSGSNDASANAGI GTWHSNSKT... SSGASVRLTC LRISGLQPEDEADYYCHuman Vλ6 germline sequences (SEQ ID NOs. 227) Vλ6 FW1 CDR1 FW2 CDR2 FW3CDR3 IGLV6-57 NFMLTQPHSVSE TPSSGS..IASNYVQ WYQQROGSSOTTVIY EDNQR.....PSGVPDRFSGSIDSSSNSAS QSYESSN..... SPGKTVTISC LTISGLKTEDEADYYCHuman Vλ7 germline sequence (from top to bottom, SEQ ID NOs. 228-229):Vλ7 FW1 CDR1 FW2 CDR2 FW3 CDR3 IGLV7-43 QTVVTQEPSLTV ASSTGAV.TSGYYPNWFQQKPGQAPRALIY STSNK.....HS WTPARFSGSL..LGGKAA LLYYGGAQ.... SPGGTVTLTCLTLSGVQPEDEAEYYC IGLV7-45 QAVVTQEPSLTV GSSTGAV.TSGHYPY WFQQKPGQAPRTLIYDTSNK.....HS WTPARFSGSL..LGGKAA LLSYSGAR.... SPGGTVTLTC LTLSGAQPEDEAEYYCHuman Vλ8 germline sequence (SEQ ID NO: 230) VλD FW1 CDR1 FW2 CDR2 FW3CDR3 IGLV8-61 QTVVTQEPSFSV GLSSGSV.STSYYPS WFQQTPGQAPRTLIY STNTR.....SSGVPDRFSGSI..LGNKAA VLYMGSGI.... SPGGTVTLTC LTITGAQADDESDYYCHuman Vλ9 germline sequence (SEQ ID NO: 231) Vλ9 FW1 CDR1 FW2 CDR2 FW3CDR3 IGLV9-49 QPVLTQPPSASA TLSSG...YSNYKVD WQQQRPGKGPRFMVR VGTGGIVGSKGDGIPDRFSVLG..SGLNRY GADHGSGSNFV* SLGASVTLTC LTIKNIQEEDESDYHCHuman Vλ10 germline sequence (SEQ ID NO: 232) Vλ10 FW1 CDR1 FW2 CDR2 FW3CDR3 IGLV10-54 QAGLTQPPSVSK TGNSNN..VGNQGAA WLQQHQGHPPKLLSY RNNNR.....PSGISERLSASR..SGNTAS SAWDSSLSA... GLRQTATLTC LTITGLQPEDEADYYCHuman Vλ11 germline sequence (SEQ ID NO: 233) Vλ11 FW1 CDR1 FW2 CDR2 FW3CDR3 IGLV11-55 RPVLTQPPSLSA TLSSDLS.VGGKNMF WYQQKPGSSPRLFLY HYSDSDKQL.GPGVPSRVSGSKETSSNTAF QVYESSAN.... SPGATARLPC LLISGLQPEDEADYYC

TABLE 5 Exemplary Human Germline Heavy Chain Consensus SequenceConsensus VH FW1 CDR1 FW2 CDR2 FW3 CDR3 All-VH EVQLVESGGGLVKPGFTFSSYAMX-- WVRQAPGKGLEWV GWISP--NGGSTYYADSVKG RFTISRDNSKNTLYLQMNGGSLRLSCAAS SLRAEDTAVYYCAR- All-VH+ EVQLVESGGGLVKP GFTFSSYAMXWSWVRQAPGKGLEWV GWISPKANGGSTYYADSVKG RFTISRDNSKNTLYLQMN GGSLRLSCAASSLRAEDTAVYYCARX VH1 QVQLVQSGAEVKKP GYTFTSYXXM-- WVRQAPGQGLEWVGWINP--XNGNTNYAQKEQG RVTITRDTSTSTAYMELS GASVKVSCKAS SLRSEDTAVYYCAR- VH2QVTLKESGPALVKP GFSLSTSGMGVX WIRQPPGKALEAL AXIY---WNDDKYYSXSLESRLTISKDTSKNQVVLTMT TQTLTLTCTFS NMDPVDTATYYCARX VH3 EVQLVESGGGLVQPGFTFSSYAMS-- WVRQAPGQGLEWV SVISS--DGXSTYYADSVKG RFTISRDNSKNSLYLQMNGGSLRLSCAAS SLRAEDTAVYYCARX VH3+ EVQLVESGGGLVQP GFTFSSYAMS--WVRQAPGKGLEWV SVISSKADGXSTYYADSVKG RFTISRDNSKNSLYLQMN GGSLRLSCAASSLRAEDTAVYYCARX VH4 QVQLQESGPGLVKP GGSISSGXYYS- WIRQPPGKGLEWIGYIY--YSGSTYYNPSLKS RVTISVDTSKNQESLKLS SETLSLTCAVS SVTAADTAVYYCAR- VH4+QVQLQESGPGLVKP GGSISSGXYYWS WIRQPPGKGLEWI GYIY--YSGSTYYNPSLKSRVTISVDTSKNQESLKLS SETLSLTCVAS SVTAADTAVYYCAR- VH5 EVQLVQSGAEVKKPGYSFTSYWIX-- WVRQMPGKGLEWS GXIYP--GDSDTRYSPSEQG HVTISADKSISTAYLQNSGESLRISCKGS SLKASDTAMYYCAR- VH6 QVQLQQSGPGLVKP GDSVSSNSAAWNWIRQSPSRGLEWL GRTYYR-SKWYNDYAVSVKS RITINPDTSKNQESLQIN SQTLSLTCAISSVTPEDTAVYYCAR- VH7 QVQLVQSGXEXRXP GYXFTXYXMN-- WVXQAPGQGLEWSGWXNT--XTGNPTYAQGETG REVESXDTSXSTAYLQIX GASVKVSCKAS SLKAEDXAXYYCAR-+Consensus sequence where consensus gaps are replaced with most commonamino acid From top to bottom: SEQ ID NOs. 234-244.

TABLE 6 Exemplary Human Germline Light Chain Consensus Sequence Vλgermline consensus sequences Consensus Vλ FW1 CDR1 FW2 CDR2 FW3 CDR3All-VL QSVLTQPPSV TGSSS--GGSYYVS WYQQKPGQAPKLLIY EDSNR-----PSGVPDRFSGSK--SGNTAS QSWDSSAXF--- SVSPGQSVTITC LTISGLQAEDEADYYC All-VL+QSVLTQPPSV TGSSSDVGGSYYVS WYQQKPGQAPKLLIY EDSNRXRXQKPSGVPDRFSGSKDASGNTAS QSWDSSAXFXXX SVSPGQSVTITC LTISGLQAEDEADYYC VL1QSVLTQPPSV SGSSSN-INGNNXVX WYQQLPGTAPKLLIY GNNXR-----PSGVPDRFSGSK--SGTSAS AAWDDSLXG--- SGAPGQRVTISC LAIIGLQSEDEADYYC VL1+QSVLTQPPSV SGSSSNIIGNNXVX WYQQLPGTAPKLLIY GNNXR-----PSGVPDRFSGSK--SGTSAS AAWDDSLXG--- SGAPGQRVTISC LAIIGLQSEDEADYYC VL2QSALTQPPSV TGTSSDVGGYNYVS WYQQHPGKAPKIMIY EVSNR-----PSGVPDRFSGSK--SGNTAS SSYAGSYTF--- SGSPGQSVTISC LTISGLQAEDEADYYC VL3SYELTQPPSV SGDX---LGXKYAH WYQQKPGQAPVLVIY KDSER-----PSGIPERFSGSS--SGNTAT QSWDSSG----- SVSPGQTARITC LTISGXQAEDEADYYC VL3+SYELTQPPSV SGDX---LGXKYAH WYQQKPGQAPVLVIY KDSER-----PSGIPERFSGSS--SGNTAT QSWDSSGXH--- SVSPGQTARITC LTISGXQAEDEADYYC VL4QPVLTQSPSA TLSSG--HSSYXIA WHQQQPGKXPRYLMK LXSDGSHSK-GDGIPDRFSGSS--SGADRY XIWXTXX----- SASLGASVRLTC LTISNLQSEDEADYYC VL4+QPVLTQSPSA TLSSG--HSSYXIA WHQQQPGKXPRYLMK LXSDGSHSK-GDGIPDRFSGSS--SGADRY XIWXTXXGQVG- SASLGASVRLTC LTISNLQSEDEADYYC VL5QPVLTQPTSL TLRSGINVGXYRIY WYQQKPGSPPRYLLX YXSDSDKXQ-GSGVPSRFSGSKDASANAGI MIWHSSAS---- SASPGASARLTC LLISGLQSEDEADYYC VL5+QPVLTQPTSL TLRSGINVGXYRIY WYQQKPGSPPRYLLX YXSDSDKXQ-GSGVPSRFSGSKDASANAGI MIWHSSASI--- SASPGASARLTC LLISGLQSEDEADYYC VL6NFMLTQPKSV TRSSGS-IASNYVQ WYQQRPGSSPTTVIY EDNQR-----PSGVPDRFSGSIDSSSNSAS QSYDSSN----- SESPGRTVTISC LTISGLKTEDEADYYC VL7QXVVTQEPSL XSSTGAVTSGXYPX WFQQKPGQAPRXLIY NISNK-----HSWIPARFSGSL--LGGKAA LLXYXGAX---- IVSPGGTVTLTC LTISGXQPEDEAEYYC VL8QTVVTQEPSF GLSSGSVSTSYYPS WYQQTPGQAPRTLIY STNTR-----SSGVPDRFSGSI--LGNKAA VLYXGSGI---- SVSPGGTVTLTC LTITGAQADDESDYYC VL9QPVLTQPPSA TLSSG--YSNYRVD WYQQRPGKGPRFVMR VGTGGTVGSRGDGIPDRFSVLG--SGLNRY GADHGSGSNFV- SASLGASVTLTC LTIKNIQEEDESDYHC VL10QAGLTQPPSV TGNSNN-VGNQAA WLQQHQGHPPKLLSY RNDDNR-----PSGISERLSASR--SGNTAS SAWDSSLSA--- SKGLRQTATLTC LTITGLQPEDEADYYC VL11RPVLTQPPSL TLSSDLSVGGKNMF WYQQKPGSSPRLFLY HYSDSDKQL-GPGVPSRVSGSKETSSNTAF QVYESSAN---- SASPGATARLPC LLISGLQPEDEADYYC+Consensus sequence where consensus gaps are replaced with most common amino acidFrom top to bottom: SEQ ID NOs. 245-261. VK germline consensus sequencesConsensus VK FW1 CDR1 FW2 CDR2 FW3 CDR3 All-VK DIVMTQSPSSRASQ------GISSYLA WYQQKPGQAPKLLIY AASSRAS GVPSRFSGSGSGTDFTLT QQYNSYPLSASPGERATISC ISSLQPEDFAVYYC All-VK+ DIVMTQSPSS RASQSLLHSDGISSYLAWYQQKPGQAPKLLIY AASSRAS GVPSRFSGSGSGTDFTLT QQYNSYP LSASPGERATISCISSLQPEDFAVYYC VK1 DIQMTQSPSS RASQ------GISXYLA WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLT QQYNSYP LSASVGDRVTITC ISSLQPEDFATYYC VK1D DIQMTQSPSSRASQ------GISSYLA WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLT QQYNSYPLSASVGDRVTITC ISSLQPEDFATYYC VK2 DIVMTQTPLS RSSQSLLHSD-GNTYLDWYLQKPGQSPQLLIY XXSNRXS GVPDRFSGSGSGTDFTLK MQATQFP LPVTXGEPASISCISRVEAEDVGVYYC VK2+ DIVMTQTPLS RSSQSLLHSDDGNTYLD WYLQKPGQSPQLLIY XXSNRXSGVPDRFSGSGSGTDFTLK MQATQFP LPVTXGEPASISC ISRVEAEDVGVYYC VK2D DIVMTQTPLSRSSQSLLHS-DGXTYLX WYLQKPGQSPQLLIY XVSNRFS GVPDRFSGSGSGTDFTLK MQATQFPLPVTPGEPASISC ISRVEAEDVGVYYC VK2D+ DIVMTQTPLS RSSQSLLHSDDGXTYLXWYLQKPGQSPQLLIY XVSNRFS GVPDRFSGSGSGTDFTLK MQATQFP LPVTPGEPASISCISRVEAEDVGVYYC VK3 EIVLTQSPAT RASQS-----VVSSYLA WYQQKPGQAPRLLIY GASTRATGIPARFSGSGSGTDFTLT QQRSNWP LSLSPGERATLSC ISSLEPEDFAVYYC VK3D EIVXTQSPATRASQS-----VXSSYLA AWQQKPGQAPRLLIY XASTRAT GIPARFSGSGSGTDFTLT QQYXNWPLSLSPGERATLSC ISSLXPEDFAVYYC VK4 DIVMTQSPDS RSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIY RASTRES GVPDRFSGSGSGTDFTLT QQYYSTP LAVSLGERATINCISSLQAEDVAVYYV VK5 XIVLTQSPXF RASX------SIGXSLH WYQQKPDQSPKLLIK YASQSFSGVPSRFSGSGSGTDFTLT HQSSSLP XSVTPXEKVTITC INSLEAEDAATYYC VK5+ XIVLTQSPXFRASXSVSF--SIGXSLH WYQQKPDQSPKLLIK YASQSFS GVPSRFSGSGSGTDFTLT HQSSSLPXSVTPXEKVTITC INSLEAEDAATYYC VK5 EIVLTQSPDF RASQ------SIGSSLHWYQQKPDQSPKLLIK YASQSFS GVPSRFSGSGSGTDFTLT HQSSSLP QSVTPKEKVTITCINSLEAEDAATYYC VK5D XXVXTQSPXF XASX------XIGXXLX WYQQKPDQXPKLLIK YASQSXSGVPSRFSGSGSGTDFTXT XQXXXXP XSVTPXEKVTITC IXSLEAEDAATYYC VK7 DIVLTQSPASRASESVS--FLGINLIH WYQQKPGQPPKLLIY QASNRDT GVPARFSGSGSGTDFTLT LQSKNFPLAVSPGQRATITC INPVEANDTANYYC+Consensus sequence where consensus gaps are replaced with most common amino acidFrom top to bottom: SEQ ID NOs. 262-277.

TABLE 7 Sequence alignment of CDR-humanized anti-A33 antibodiesA33 Rabbit ELVMTQTPPSLSASVGETVRIRC L AS EFLFNGVS WYQQKPGKPPKFLIS G AS NL E SGVPP RFSGSGSGTDYTLTIGGVQAEDVATYYC LGG YS GSSG LTFGAGTNVEIK DPK9/JK4DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYS  TPLTFGGGTKVEIK ABS-A33-1DIQMTQSPSSLSASVGDRVTITCRASQSISSY VS WYQQKPGKAPKLLIY G AS N LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LGG YSGST G LTFGGGTKVEIK ABS-A33-2DIQMTQSPSSLSASVGDRVTITCRAS EFLFNGVS WYQQKPGKAPKLLIY G ASSL E SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGS SG LTFGGGTKVEIK ABS-A33-3DIQMTQSPSSLSASVGDRVTITCRAS E SLSSYL S WYQQKPGKAPKLLIY G AS N LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LGG YSGS SG LTFGGGTKVEIK ABS-A33-4DIQMTQSPSSLSASVGDRVTITCRAS QFLFN GL S WYQQKPGKAPKLLIY G AS N LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGST G LTFGGGTKVEIK ABS-A33-6DIQMTQSPSSLSASVGDRVTITCRAS QFLFNGVS WYQQKPGKAPKLLIYAAS N L E SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGST G LTFGGGTKVEIK ABS-A33-7DIQMTQSPSSLSASVGDRVTITCRAS EFLFNGVS WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGS SG LTFGGGTKVEIK ABS-A33-8DIQMTQSPSSLSASVGDRVTITCRAS QFLFNGVS WYQQKPGKAPKLLIYAAS N LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGS SG LTFGGGTKVEIK ABS-A33-9DIQMTQSPSSLSASVGDRVTITCRAS QFLFN VLVWYQQKPGKAPKLLIYAAS N L E SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGST G LTFGGGTKVEIK ABS-A33-10DIQMTQSPSSLSASVGDRVTITCRAS EFLFNGVS WYQQKPGKAPKLLIY G ASSL E SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGS SG LTFGGGTKVEIK ABS-A33-11DIQMTQSPSSLSASVGDRVTITCRAS EFLFNGVS WYQQKPGKAPKLLIY G ASSL E SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGG YSGS SG LTFGGGTKVEIK A33 RabbitQEQLMESGGGLVTLGGSLKLSCKASG ID FS H Y GI SWVRQAPGKGLEWIA Y I YPNY GS VDYA SW V N G RFTISLDNAQNTVFLQMISLTAADTATYFCAR DRGYYSGSRGTRLDL WGQGTLVTISSDP47/JH4EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK               WGQGTLVTVSS ABS-A33-1EVQLLESGGGLVQPGGSLRLSCAASGF D FSSYAMSWVRQAPGKGLEWVSAIS PN GGSVYYADSV N GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGYYTGSRGTRLALWGQGTLVTVSS ABS-A33-2EVQLLESGGGLVQPGGSLRLSCAASGFTFS H Y GI SWVRQAPGKGLEWVS Y I YPNY GST DYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQYYSGSRGTRLDLWGQGTLVTVSSABS-A33-3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAISWVRQAPGKGLEWVS Y I Y G NGGS VD YA SW V N GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRVYYSVSRGTRLDLWGQGTLVTVSS ABS-A33-4EVQLLESGGGLVQPGGSLRLSCAASGF D FS H Y GI SWVRQAPGKGLEWVS Y I YP S Y GST DYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGYYSGSRGTRLDLWGQGTLVTVSSABS-A33-6 EVQLLESGGGLVQPGGSLRLSCAASGFTFS H Y GI SWVRQAPGKGLEWVS Y I YP SY GST D YASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGAYSGSRGTRLDLWGQGTLVTVSS ABS-A33-7EVQLLESGGGLVQPGGSLRLSCAASG ID FSSY GI SWVRQAPGKGLEWVS Y I YP S Y GST DYAD W VKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGYYSGSRGTRLDLWGQGTLVTVSSABS-A33-8 EVQLLESGGGLVQPGGSLRLSCAASG ID FSSY GI SWVRQAPGKGLEWVS Y I YP SY GST D YA SW V N GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGVYSGSRGTRLDLWGQGTLVTVSS ABS-A33-9EVQLLESGGGLVQPGGSLRLSCAASG ID FS H Y GI SWVRQAPGKGLEWVS Y I YPNY GST DYA SW V N G RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDKGYYSGSRGTRLDLWGQGTLVTVSSABS-A33-10 EVQLLESGGGLVQPGGSLRLSCAASGFTFS H YG ISWVRQAPGKGLEWVS Y I YPNYGST D YADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQYYSGSRGTRLDLWGQGTLVTVSSA33 Rabbit = rabbit anti-A33 v-gene sequences. CDRs in bold. From Top toBottom: SEQ ID NOs. 353-375.

TABLE 8 Sequence alignment of CDR-humanized antib-pTau antibodiespT231 pTau   ALTQPTSVSANLGGSVEITC S G SD -- YD Y G -WYQQKAPGSAPVTVIYWNDK RPSDIPS RFSGSTSGSTSTLTITGVQAEDEAVYYC GAY DGS AGGGI FGAGTTLTVLDPL16/Jλ2 SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQK-PGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL C11-ABS-pTAUSSELTQDPAVSVALGQTVRITCQGD D --SYY G -WYQQK-PGQAPVTVIYG N NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DGSG G H G VFGGGTKLTVL C9-ABS-pTAUSSELTQDPAVSVALGQTVRITC S GD D --SYY G -WYQQK-PGQAPVTVIYG N NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DGSG G H G VFGGGTKLTVL C18-ABS-pTAUSSELTQDPAVSVALGQTVRITCQGD D --SYY G -WYQQK-PGQAPVTVIYG NDK RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DGSG GGG VFGGGTKLTVL C4-ABS-pTAUSSELTQDPAVSVALGHTVRITC S GD D --SYY G -WYQQK-PGQAPVTVIYG N NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DSSG GGGI FGGGTKLTVL C13-ABS-pTAUSSELTQDPAVSVALGQTVRITCQGD D --SYY G -WYQQK-PGQAPVTVIYG N N K RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DSSG GGG VFGGGTKLTVL C21-ABS-pTAUSSELTQDPAVSVALGQTVRITCQGD D --SYY G -WYQQK-PGQAPVTVIYG ND NRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DSSG GGGI FGGGTKLTVL C6-ABS-pTAUSSELTQDPAVSVALGQTVRITCQGD D --SYY G -WYQQK-PGQAPVTVIYG ND NRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC GAY DSSG GGGI FGGGTKLTVL C8-ABS-pTAUSSELTQDPAVSVALGQTVRITCQG SD -- Y YY G -WYQQK-PGQAPVTVIYG ND NRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYC G S Y DSS AG H G VFGGGTKLTVL pT231 pTauAVTLDESGGGLQTPGGGLSLVCKASGFT L SSY Q M M WVRQAPGKGLEWVAGITSRGGVTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAK PALDSDQCGFPEAGC ID A WGHGTEVIVSSDP47/JH4EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK               FDYWGQGTLVTVSSC11-ABS-pTAU EVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q M MWVRQAPGKGLEWVAGITSRGGVTGYGDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPALDSDQCGFPEAGCI D A WGQGTLVTVSS C09-ABS-pTAUEVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q M M WVRQAPGKGLEWVAGITSRGGVTGYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PALDADQCGFPEAGCI D A WGQGTLVTVSSC18-ABS-pTAU EVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q M MWVRQAPGKGLEWVAGITSRGGVTGYGSAVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPALDSDQCGFPEAGCI D S WGQGTLVTVSS C04-ABS-pTAUEVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q MSWVRQAPGKGLEWVAGITSRGGVTGYGDAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PALDADQCGFPEAGCI D A WGQGTLVTVSSC13-ABS-pTAU EVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q M MWVRQAPGKGLEWVSGITSRGGVTGYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPALDSDQCGFPEAGCI D A WGQGTLVTVSS C21-ABS-pTAUEVQLLESGGGLVQPGGSLRLSCKASGFT L SSY Q M M WVRQAPGKGLEWVAGITSRGGVTGYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PALDSDQCGFPEAGCI D A WGQGTLVTVSSC06-ABS-pTAU EVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q M MWVRQAPGKGLEWVSGITSRGGVTGYGSSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPALNSDQCGFPEAGCI D A WGQGTLVTVSS C08-ABS-pTAUEVQLLESGGGLVQPGGSLRLSCKASGFTFSSY Q MSWVRQAPGKGLEWVSGITGRGGVTGYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PALDSDQCGFPEAGCI D A WGQGTLVTVSS FromTop to Bottom: SEQ ID NOs. 376-395.

TABLE 9 Sequence alignment of CDR-humanized anti-RAGE antibodiesXTM4 RAGE DIQMTQSPSSMSVSLGDTITITCRASQ DVGI Y V NWFQQKPGKSPRRMIY R A TN LAD GVPSRFSGSRSGSIYS LTISSLESEDVADYHC LEFDEH PLTFGSGTKVEIK DPK9/JK4DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGYPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK C4-ABS-RAGEDIQMTQSPSSLSASVGDRVTITCRASQS VG SY V NWYQQKPGKAPKLLIY R ASSL ADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYC LEFDEH PLTFGGGTKVEIK C11-ABS-RAGEDIQMTQSPSSLSASVGDRVTITCRASQS VG SY V NWYQQKPGKAPKLLIY R AS NLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYC LEFDEH PLTFGGGTKVEIK C10-ABS-RAGEDIQMTQSPSSLSASVGDRVTITCRASQ DVG SY V NWYQQKPGKAPKLLIY R AS N L ADGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQ EF YS H PLTFGGGTKVEIK C3-ABS-RAGEDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY R A TNLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYC LEFDEH PLTFGGGTKVEIK C7-ABS-RAGEDIQMTQSPSSLSASVGDRVTITCRASQSI G SYLNWYQQKPGKAPKLLIY R ASSL ASGVPSRFSGSGSGTD FTLTISSLQPEDFATYYC LEFDEH PLTFGGGTKVEIK XTM4 RAGEEVQLVESGGGLVQPGRSLKLSCVVSGFTF NN YWM T WIRQTPGKGLEWVA S I DNS G DNT YY PDSVK D RFTIS RDNAKSTLYLQMNSLRSEDTATYYCTR GGDITTGF DYWGQGVMVTVSS DP54/JH4EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR        DYWGQGTLVTVSS C4-ABS-RAGEEVQLVESGGGLVQPGGSLRLSCAASGFTFS N YWMSWVRQAPGKGLEWVA S IKNDGD NTYYVDSVKDRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCAR GGDITTGLD YWGQGTLVTVSSC11-ABS-RAGE EVQLVESGGGLVQPGGSLRLSCAAS GFTFS N YWMSWVRQAPGKGLEWVAT S IDNS GS N KYYVDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCAR GGDITTGFDYWGQGTLVTVSS C10-ABS-RAGEEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA S IK NS GSE T YY PDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCAR GGDITTGF DYWGQGTLVTVSSC3-ABS-RAGE EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA S I DQDGSEKYY P DSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCAR GGDITTGL DYWGQGTLVTVSSC7-ABS-RAGE EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA S I D QDGSN KYY P DSVKGRFT ISRDNAKNSLYLQMNSLRAEDTAVYYCAR GGDITTGL DYWGQGTLVTVSSXTM4 RAGE = rat anti-RAGE v-gene sequences. From Top to Bottom: SEQ IDNOs. 396-409.

What is claimed is:
 1. A humanized antibody or antigen-binding fragmentthereof that binds to a target antigen, wherein: (a) said antibody orantigen-binding fragment thereof comprises (i) a VH domain comprising: ahuman germline VH framework sequence, and CDR-H1, CDR-H2, and CDR-H3;and (ii) a VL domain comprising: a human germline VL framework sequence,and CDR-L1, CDR-L2, and CDR-L3; (b) said CDR-L1, CDR-L2, CDR-L3, CDR-H1,and CDR-H2 are derived from corresponding CDRs from a non-human donorantibody that binds to said target antigen; (c) for each position withinsaid CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, the residue is eitherhuman germline residue from said human germline VL or VH, orcorresponding residue from said non-human donor antibody; (d) saidCDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each comprises at least onemore human germline residue as compared to the corresponding non-humandonor CDR, (e) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 eachcomprises at least one more non-human donor residue as compared to thecorresponding human germline VH or VL CDR; and (f) for each positionwithin CDR-H3, the residue is any one of the 20 natural amino acidresidues.
 2. The antibody or antigen-binding fragment thereof of claim1, wherein said human germline VH framework sequence comprises a VH3,VH1, or VH5 framework sequence.
 3. The antibody or antigen-bindingfragment thereof of claim 1, wherein said human germline VH frameworksequence comprises a VH germline consensus framework sequence.
 4. Theantibody or antigen-binding fragment thereof of claim 1, wherein saidhuman germline VH framework sequence comprises the VH framework sequenceof any one of the consensus sequences listed in Table 2 and Table
 5. 5.The antibody or antigen-binding fragment thereof of claim 1, whereinsaid human germline VL framework sequence comprises a V_(K) or V_(λ)framework sequence.
 6. The antibody or antigen-binding fragment thereofof claim 1, wherein said human germline VL framework sequence comprisesa VL germline consensus framework sequence.
 7. The antibody orantigen-binding fragment thereof of claim 1, wherein said human germlineVL framework sequence comprises the VL framework sequence of any one ofthe human germline sequences listed in Table 4 and Table
 6. 8. A methodof generating a library comprising a plurality of polypeptides, forselection of a humanized antibody that binds to a target antigen,comprising: (a) obtaining the sequence a non-human donor antibody thatbinds to said target antigen, and determining the donor CDR-L1, CDR-L2,CDR-L3, CDR-H1, and CDR-H2 sequences of said non-human antibody; (b)obtaining the sequences of a human germline VL and a human germline VH,and determining the germline framework and germline CDR-L1, CDR-L2,CDR-L3, CDR-H1, and CDR-H2 sequences of said human VL and VH; (c)aligning each of the non-human donor CDR-L1, CDR-L2, and CDR-L3sequences with the corresponding germline CDR sequence from said humanVL, and each of the non-human CDR-H1 and CDR-H2 sequences withcorresponding germline CDR sequence from said human VH; (d) identifyingpositions in CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 where humangermline residue is the same as, or different from, the correspondingnon-human donor residue; (e) generating a library of polypeptides, eachpolypeptide comprising an antibody variable domain, wherein saidantibody variable domain comprises (1) a VH domain comprising: theframework sequence of the human germline VH from step (b), and CDR-H1,CDR-H2, and CDR-H3; and (2) a VL domain comprising: the frameworksequence of the human germline VL from step (b), and CDR-L1, CDR-L2, andCDR-L3; wherein: (i) for each individual position within CDR-L1, CDR-L2,CDR-L3, CDR-H1 and CDR-H2: if the human germline residue at saidposition is the same as the corresponding non-human donor residue, allpolypeptides in the library comprise the human germline residue at saidposition; if the human germline residue at the position is differentfrom the corresponding non-human donor residue, a portion of thepolypeptides in the library comprise the human germline residue at saidposition, the remainder of the polypeptides comprise the correspondingnon-human donor residue at said position; (ii) for each individualposition within CDR-H3, the residue is any one of the 20 natural aminoacid residues; (iii) less than 1% of the polypeptides in said librarycomprise the original non-human donor CDR-L1, CDR-L2, CDR-L3, CDR-H1,and CDR-H2 sequences; and (iv) less than 1% of the polypeptides in saidlibrary comprise the original human VL germline CDR-L1, CDR-L2, andCDR-L3, and the original human VH germline CDR-H1 and CDR-H2 sequences.9. The method of claim 8, wherein for each individual position withinCDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, if the human germlineresidue and non-human donor residue are different according to step (d),the percentage of polypeptides comprising the human germline residue atsaid position is from 15% to 85%, the remainder comprising thecorresponding non-human donor residue at said position.
 10. The methodof claim 8, wherein for each individual position within CDR-H3, each ofthe 20 natural amino acid residues is represented by at least 0.1% ofthe polypeptides in the library.
 11. The method of claim 8, wherein saidhuman germline VH framework sequence comprises a VH3, VH1, or VH5framework sequence.
 12. The method of claim 8, wherein said humangermline VH framework sequence comprises a VH germline consensusframework sequence.
 13. The method of claim 8, wherein said humangermline VH framework sequence comprises the VH framework sequence ofany one of the consensus sequences listed in Table 2 and Table
 5. 14.The method of claim 8, wherein said human germline VL framework sequencecomprises a V_(K) or V_(λ) framework sequence.
 15. The method of claim8, wherein said human germline VL framework sequence comprises a VLgermline consensus framework sequence.
 16. The method of claim 8,wherein said human germline VL framework sequence comprises the VLframework sequence of any one of the human germline sequences listed inTable 4 and Table
 6. 17. A library for humanizing a non-human donorantibody that binds to a target antigen, wherein: (a) said librarycomprises a plurality of polypeptides, each polypeptide comprising anantibody variable domain; (b) said antibody variable domain comprises(i) a VH domain comprising: a human germline VH framework sequence, andCDR-H1, CDR-H2, and CDR-H3; and (ii) a VL domain comprising: a humangermline VL framework sequence, and CDR-L1, CDR-L2, and CDR-L3; (c) foreach individual position within said CDR-L1, CDR-L2, CDR-L3, CDR-H1, andCDR-H2, if the human germline residue at said position is the same asthe corresponding non-human donor residue, all polypeptides in thelibrary comprise the human germline residue at said position; if thehuman germline residue at the position is different from thecorresponding non-human donor residue, a portion of the polypeptides inthe library comprise the human germline residue at said position, theremainder of the polypeptides comprise the corresponding non-human donorresidue at said position; (d) for each individual position withinCDR-H3, the residue is any one of the 20 natural amino acid residues;(e) less than 1% of the polypeptides in said library comprise theoriginal non-human donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2sequences; and (f) less than 1% of the polypeptides in said librarycomprise the original human VL germline CDR-L1, CDR-L2, and CDR-L3, andthe original human VH germline CDR-H1 and CDR-H2 sequences.
 18. Thelibrary of claim 17, wherein for each individual position within CDR-L1,CDR-L2, CDR-L3, CDR-H1, and CDR-H2, if the human germline residue andnon-human donor residue are different according to (c), the percentageof polypeptides comprising the human germline residue at said positionis from 15% to 85%, the remainder comprising the corresponding non-humandonor residue at said position.
 19. The library of claim 17, wherein foreach individual position within CDR-H3, each of the 20 natural aminoacid residues is represented by at least 0.1% of the polypeptides in thelibrary.
 20. The library of claim 17, wherein said human germline VHframework sequence comprises a VH3, VH1, or VH5 framework sequence. 21.The library of claim 17, wherein said human germline VH frameworksequence comprises a VH germline consensus framework sequence.
 22. Thelibrary of claim 17, wherein said human germline VH framework sequencecomprises the VH framework sequence of any one of the consensussequences listed in Table 2 and Table
 5. 23. The library of claim 17,wherein said human germline VL framework sequence comprises a V_(K) orV_(λ) framework sequence.
 24. The library of claim 17, wherein saidhuman germline VL framework sequence comprises a VL germline consensusframework sequence.
 25. The library of claim 17, wherein said humangermline VL framework sequence comprises the VL framework sequence ofany one of the human germline sequences listed in Table 4 and Table 6.